Modified Vip3 polypeptides

ABSTRACT

The present invention is directed to vegetative insecticidal proteins (Vips) modified to comprise heterologous carbohydrate binding modules and methods of use thereof.

RELATED APPLICATION INFORMATION

This application is a 371 of International Application No.PCT/US2015/047071, filed 27 Aug. 2015, which claims priority to U.S.Provisional Application No. 62/043,922, filed 29 Aug. 2014, the contentsof which are incorporated herein by reference herein.

STATEMENT REGARDING ELECTRONIC FILING OF A SEQUENCE LISTING

A Sequence Listing in ASCII text format, submitted under 37 C.F.R. §1.821, entitled 9207-116WO_ST25.txt, 341,238 bytes in size, generatedAug. 25, 2015, and filed via EFS-Web, is provided in lieu of a papercopy. This Sequence Listing is hereby incorporated herein by referenceinto the specification for its disclosures.

FIELD OF THE INVENTION

The invention relates to vegetative insecticidal proteins (Vip) modifiedto comprise heterologous carbohydrate binding modules and methods of usethereof.

BACKGROUND OF THE INVENTION

Bacillus thuringiensis (Bt) are ubiquitous soil dwelling, gram positivespore-forming bacteria. Bt produces protein toxins which are orallyactive and highly specific for individual insect orders and species (K.van Frankenhuyzen, J. Invertebr. Pathol. 101, 1-16 (2009)). Thus, Btproteins and the bacilli that produce them have been utilized inagriculture since the 1920s for control of insect pests (J. Lord, JInvertebr Pathol. 89, 19-29 (2005)). To ease field application and totarget plant tissues not readily protected by foliar application, selectproteins have been transgenically expressed in crops widely since the1990s.

Bt produces three known classes of insecticidal protein toxins: crystal(Cry), cytolytic (Cyt), and vegetative insecticidal proteins (Vip). Cryproteins are produced as parasporal intracellular inclusion bodies withmicroscopic crystal morphology. Cyt proteins do not share sequencehomology with the Cry proteins but are similarly produced as inclusionbodies during sporulation. Vip proteins are soluble toxins from Bt whichare produced throughout the vegetative life cycle of the bacteria (A.Bravo et al. Insect Biochem Mol Biol. 41(7):423-31(2011)).

Biological pest control agents, such as Bacillus thuringiensis strainsexpressing pesticidal polypeptides have been applied to crop plants withsatisfactory results, thus offering an alternative or compliment tochemical pesticides. The expression of Cry proteins in transgenic plantshas provided efficient protection against certain insect pests, andtransgenic plants expressing such proteins have been commercialized,allowing farmers to reduce or eliminate applications of chemical insectcontrol agents.

Vip3 is a specific class of vegetative insecticidal protein, which hasbroad toxicity against lepidopteran pest species and is amenable totransgenic plant expression (J. Estruch et al. Proc Natl Acad Sci USA93, 5389-94 (1996)). The first product containing Vip3 was geneticallymodified corn sold under the brand name AGRISURE VIPTERA™ by Syngenta in2011 (See also Syngenta U.S. Pat. Nos. 7,378,493 and 7,244,820).Nevertheless, compared to the vast peer reviewed literature on the Cryproteins, relatively little is reported for the Vip3 proteins. Vip3proteins share no homology with Cry or Cyt proteins. Vip3 does not BLASTto any other confirmed proteins in the nr protein database with expectvalues less than 1.0. Currently reported sequences indicate far lesssequence variation between the Vip3 proteins compared to variationobserved for the Cry proteins.

Vip3 proteins are approximately 88 kDa in size and are produced andsecreted by Bacillus during its vegetative stage of growth (vegetativeinsecticidal proteins, Vip). The Vip3A protein possesses insecticidalactivity against a wide spectrum of lepidopteran pests, including, butnot limited to, black cutworm (ICW, Agrotis ipsilon), fall armyworm(FAW, Spodoptera frugiperda), tobacco budworm (TBW, Heliothisvirescens), and corn earworm (CEW, Helicoverpa zea), but has no activityagainst the European corn borer (ECB, Ostrinia nubilalis). Thus, theVip3A protein displays a unique spectrum of insecticidal activities.More recently, plants expressing the Vip3A protein have been found to beresistant to feeding damage caused by hemipteran insect pests (U.S. Pat.No. 6,429,360). Additional members of the Vip3 class of proteins havebeen identified (see, e.g., WO03/075655, WO02/078437, WO 98/18932, WO98/33991, WO 98/00546, and WO 99/57282).

Numerous commercially valuable plants, including common agriculturalcrops, are susceptible to attack by insect pests, causing substantialreductions in crop yield and quality. For example, growers of maize (Zeamays), face a major problem with combating pest infestations. Insects,including Lepidopteran and Coleopteran insects, annually destroy anestimated 15% of agricultural crops in the United States and an evengreater percentage in developing countries. In addition, competitionwith weeds and parasitic and saprophytic plants account for even morepotential yield losses. Yearly, such pests cause over $100 billion incrop damage in the United States alone.

In an effort to combat pest infestations, various methods have beenemployed in order to reduce or eliminate pests in a particular plot.These efforts include rotating corn with other crops that are not a hostfor a particular pest and applying pesticides to the above-groundportion of the crop, applying pesticides to the soil in and around theroot systems of the affected crop. Traditionally, farmers have reliedheavily on chemical pesticides to combat pest damage.

There is a demand for alternative insecticidal agents for agriculturalcrops. For example, maize plants incorporating transgenic genes whichcause the maize plant to produce insecticidal proteins providingprotection against target pest(s) is another approach to controllingpests. Therefore, there remains a need to discover new and effectivepest control agents that provide an economic benefit to farmers.Particularly needed are control agents that are targeted to a widerspectrum of economically important insect pests and that have a highspecific activity against insect pests that are or could becomeresistant to existing insect control agents.

SUMMARY OF THE INVENTION

In some embodiments, a modified Vip3 polypeptide comprising aheterologous carbohydrate binding module (CBM) is provided. In someaspects, the heterologous CBM is substituted for all or a portion ofDomain III of a Vip3 polypeptide. In some embodiments, the modified Vip3polypeptide comprises all or a portion of Domain I and/or Domain II of aVip3 polypeptide. In some embodiments, the modified Vip3 polypeptidecomprises all or a portion of Domain IV of a Vip3 polypeptide oralternatively, lacks all or a portion of Domain IV of a Vip3polypeptide. In some embodiments, the modified Vip3 polypeptide ispesticidal against, for example, insects, such as, for example, a fallarmyworm. In some embodiments, a modified Vip3 polypeptide as describedherein demonstrates insecticidal activity against a Vip3 resistant fallarmyworm colony, such as, for example, a Vip3A resistant fall armywormcolony.

In another aspect, a composition is provided, the composition comprisinga modified Vip3 polypeptide of the invention in an agriculturallyacceptable carrier.

In some embodiments, the invention provides nucleic acid moleculesand/or nucleotide sequences encoding modified Vip3 polypeptides of theinvention and expression cassettes and recombinant vector comprising anucleic acid molecule and/or nucleotide sequences encoding modified Vip3polypeptides of the invention.

In further aspects, an extract from a transgenic seed or a transgenicplant of the invention is provided, wherein the extract comprises anucleic acid molecule and/or a modified Vip3 polypeptide of theinvention. Thus, in some embodiments, a composition comprising saidextract is provided. In a further embodiment, the composition maycomprise said extract in an agriculturally acceptable carrier.

In some embodiments, a method of providing a farmer with a means ofcontrolling a plant pest is provided, the method comprising supplying tothe farmer plant material or bacteria, said plant material or bacteriacomprising a nucleic acid molecule that encodes the modified Vip3polypeptide according to the invention.

In some aspects, a method of producing a modified Vip3 polypeptide ofthe invention is provided, comprising the steps of: (a) transforming ahost cell with a recombinant nucleic acid molecule comprising anucleotide sequence encoding for the modified Vip3 polypeptide; and (b)culturing the host cell of step (a) under conditions in which the hostcell expresses the recombinant nucleic acid molecule, thereby producingthe modified Vip3 polypeptide. In some embodiments, a method ofproducing a modified Vip3 polypeptide is provided, the methodcomprising, transforming a host cell with a nucleic acid moleculecomprising a promoter operably linked to a nucleotide sequence encodingthe modified Vip3 polypeptide of the invention; growing the host cellunder conditions which allow expression of the modified Vip3polypeptide; and recovering the modified Vip3 polypeptide. In someembodiments, a method of producing a modified Vip3 polypeptide isprovided, the method comprising, growing a host cell of the inventionunder conditions which allow expression of the modified Vip3polypeptide; and recovering the modified Vip3 polypeptide.

In some embodiments, a method of reducing damage in a transgenic plantcaused by a plant pest is provided, the method comprising planting atransgenic plant seed comprising a nucleic acid molecule that expressesthe modified Vip3 polypeptide of the invention, thereby reducing damagecaused by the pest to a transgenic plant grown from the transgenic plantseed.

In some embodiments, the invention provides a method of controlling apest comprising providing the transgenic plant of the invention andapplying to the plant or the seed a crop protection product. In someembodiments, the pest is a fall armyworm.

In some embodiments, a method of controlling pests is provided, themethod comprising contacting the pests with a pesticidally effectiveamount of the composition of the invention. In some embodiments, amethod of protecting a plant and/or a plant propagation material isprovided, the method comprising contacting the plant and/or plantpropagation material with an effective amount of the composition of theinvention. In some embodiments, the method comprises a method ofcontrolling a fall armyworm colony.

In further aspects, a method of increasing pesticidal activity in aplant, plant part or plant cell is provided, the method comprisingintroducing one or more nucleic acid molecules encoding one or moremodified Vip3 polypeptides of the invention into a plant, plant part orplant cell to produce a transgenic plant, plant part or plant cell thatexpresses the one or more nucleic acid molecules, wherein the one ormore nucleic acid molecules encode for a polypeptide comprisingpesticidal activity, thereby increasing pesticidal activity in thetransgenic plant, plant part or plant cell as compared with a control.

In some embodiments, a modified Vip3 polypeptide and/or composition asdescribed herein is active and/or insecticidal against a Vip3 resistantfall armyworm colony, such as, for example, a Vip3A resistant fallarmyworm colony.

In some embodiments, transgenic host cells, including bacterial andplant cells, plants, and plant parts, including seeds, comprising anucleic acid molecule and/or nucleotide sequences encoding modified Vip3polypeptides of the invention are provided, as well as crops, andharvested and processed products produced therefrom.

These and other aspects of the invention are set forth in more detail inthe description of the invention below.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a polyacrylamide gel electrophoresis (PAGE) gel of thesoluble fraction of lysed E. coli expressing various Vip3D Domain IIICBM swaps. The samples assayed are in Table 1 are, from left to right:molecular weight ladder, 1OF3, 1PMH, 1WKY, 2BGP, 2ZEZ, CenC, gp21,PsHGF7.

FIG. 2 shows ribbon diagrams of CBM structures. Top row from left toright: Vip3D Domain III, 2ZEZ, 2BGP. Middle row: 1OF3, 2ZEX, 1PMH.Bottom: 1WKY. Calcium (depicted as spheres) is shown. Sugar ligands ofco-crystal structures are shown in their binding grooves (stickstructures). Cloning junctions on the polypeptide chain are indicated assticks.

FIG. 3 shows a sequence alignment of three domain swaps (CenC—SEQ ID NO.30, GP21—SEQ ID NO: 29, PsHGF7—SEQ ID NO. 31) that were made based onamino acid sequence similarity to 2ZEX. The sequences share 84%consensus and 8% identity.

FIG. 4 shows a sodium dodecyl sulfate (SDS) PAGE gel of soluble E. colicrude extracts of the induced Vip3A-CBM chimeras (lanes in parentheses):Vip3A (1), 2ZEX (2), 2ZEZ (3), 1OFE (4), 1PMH (5), 2GBP (6), CENC (7),GP21 (8), and PSHGF7 (9)

FIG. 5A-5C show schematics of the CBM substitutions in Vip3. FIG. 5Ashows a portion of the Vip3D P021 (SEQ ID NO. 6) sequence with aminoacid residue 541 and 668 marked, as well as the exchange of Vip3D P021amino acid residues 542 to 667 with the 2ZEX CBM (SEQ ID NO: 24) and the2ZEZ CBM (SEQ ID NO: 25); FIG. 5B shows the exchange of Vip3D P021 (SEQID NO. 6) amino acid residues 542 to 667 with the 10FE CBM (SEQ ID NO.26), the 1PMH CBM (SEQ ID NO. 27) and the 2BGP CBM (SEQ ID NO. 28); FIG.5C shows the exchange of the Vip3D P021 (SEQ ID NO. 6) amino acidresidues 542 to 667 with the GP21 CBM (SEQ ID NO. 29), the CENC CBM (SEQID NO. 30), the PsHGF7 CBM (SEQ ID NO. 31) or the 1WKY CBM (SEQ ID NO.32).

BRIEF DESCRIPTION OF SEQUENCES IN THE SEQUENCE LISTING

SEQ ID NO: 1 is the amino acid sequence of Vip3D.

SEQ ID NO:2 is the amino acid sequence of Vip3A.

SEQ ID NO:3 is the amino acid sequence of Vip3B.

SEQ ID NO:4 is the amino acid sequence of Vip3C.

SEQ ID NO:5 is a consensus amino acid sequence of Vip3.

SEQ ID NO:6 is the amino acid sequence of P021 (10His-Vip3D-AAPF).

SEQ ID NO:7 is the amino acid sequence of P021 with Domain III swap to2ZEX.

SEQ ID NO:8 is the amino acid sequence of P021 with Domain III swap to2ZEZ.

SEQ ID NO:9 is the amino acid sequence of P021 with Domain III swap to10FE.

SEQ ID NO:10 is the amino acid sequence of P021 with Domain III swap to1PMH.

SEQ ID NO:11 is the amino acid sequence of P021 with Domain III swap to28GP.

SEQ ID NO:12 is the amino acid sequence of P021 with Domain III swap toGP21.

SEQ ID NO: 13 is the amino acid sequence of P021 with Domain III swap toCenC.

SEQ ID NO:14 is the amino acid sequence of P021 with Domain III swap toPSHGF7.

SEQ ID NO:15 is the amino acid sequence of P021 with Domain III swap to1WKY.

SEQ ID NO:16 is the amino acid sequence of Vip3A with Domain III swap to2ZEX.

SEQ ID NO:17 is the amino acid sequence of Vip3A with Domain III swap to2ZEZ.

SEQ ID NO:18 is the amino acid sequence of Vip3A with Domain III swap to10FE.

SEQ ID NO:19 is the amino acid sequence of Vip3A with Domain III swap to1PMH.

SEQ ID NO:20 is the amino acid sequence of Vip3A with Domain III swap to2BGP.

SEQ ID NO:21 is the amino acid sequence of Vip3A with Domain III swap togp21.

SEQ ID NO:22 is the amino acid sequence of Vip3A with Domain III swap toCenC.

SEQ ID NO:23 is the amino acid sequence of Vip3A with Domain III swap toPsHGF7.

SEQ ID NO:24 is the amino acid sequence of the 2ZEX domain.

SEQ ID NO:25 is the amino acid sequence of the 2ZEZ domain.

SEQ ID NO:26 is the amino acid sequence of the 10FE domain.

SEQ ID NO:27 is the amino acid sequence of the 1PMH domain.

SEQ ID NO:28 is the amino acid sequence of the 2BGP domain.

SEQ ID NO:29 is the amino acid sequence of the gp21 domain.

SEQ ID NO:30 is the amino acid sequence of the CenC domain.

SEQ ID NO:31 is the amino acid sequence of the PsHGF7 domain.

SEQ ID NO:32 is the amino acid sequence of the 1WKY domain

SEQ ID NO:33 is the amino acid sequence of Vip3D with Domain III swap to2ZEX.

SEQ ID NO:34 is the amino acid sequence of Vip3D with Domain III swap to2ZEZ.

SEQ ID NO:35 is the amino acid sequence of Vip3D with Domain III swap to10FE.

SEQ ID NO:36 is the amino acid sequence of Vip3D with Domain III swap to1PMH.

SEQ ID NO:37 is the amino acid sequence of Vip3D with Domain III swap to2BGP.

SEQ ID NO:38 is the amino acid sequence of Vip3D with Domain III swap toGP21.

SEQ ID NO:39 is the amino acid sequence of Vip3D with Domain III swap toCenC.

SEQ ID NO:40 is the amino acid sequence of Vip3D with Domain III swap toPSHGF7.

SEQ ID NO:41 is the amino acid sequence of Vip3B with Domain III swap to2ZEX.

SEQ ID NO:42 is the amino acid sequence of Vip3B with Domain III swap to2ZEZ.

SEQ ID NO:43 is the amino acid sequence of Vip3B with Domain III swap to10FE.

SEQ ID NO:44 is the amino acid sequence of Vip3B with Domain III swap to1PMH.

SEQ ID NO:45 is the amino acid sequence of Vip3B with Domain III swap to2BGP.

SEQ ID NO:46 is the amino acid sequence of Vip3B with Domain III swap toGP21.

SEQ ID NO:47 is the amino acid sequence of Vip3B with Domain III swap toCenC.

SEQ ID NO:48 is the amino acid sequence of Vip3B with Domain III swap toPSHGF7.

SEQ ID NO:49 is the amino acid sequence of Vip3C with Domain III swap to2ZEX.

SEQ ID NO:50 is the amino acid sequence of Vip3C with Domain III swap to2ZEZ.

SEQ ID NO:51 is the amino acid sequence of Vip3C with Domain III swap to10FE.

SEQ ID NO:52 is the amino acid sequence of Vip3C with Domain III swap to1PMH.

SEQ ID NO:53 is the amino acid sequence of Vip3C with Domain III swap to2BGP.

SEQ ID NO:54 is the amino acid sequence of Vip3C with Domain III swap toGP21.

SEQ ID NO:55 is the amino acid sequence of Vip3C with Domain III swap toCenC.

SEQ ID NO:56 is the amino acid sequence of Vip3C with Domain III swap toPSHGF7.

DETAILED DESCRIPTION

The present invention now will be described hereinafter with referenceto the accompanying drawings and examples, in which embodiments of theinvention are shown. This description is not intended to be a detailedcatalog of all the different ways in which the invention may beimplemented, or all the features that may be added to the instantinvention. For example, features illustrated with respect to oneembodiment may be incorporated into some embodiments, and featuresillustrated with respect to a particular embodiment may be deleted fromthat embodiment. Thus, the invention contemplates that in someembodiments of the invention, any feature or combination of features setforth herein can be excluded or omitted. In addition, numerousvariations and additions to the various embodiments suggested hereinwill be apparent to those skilled in the art in light of the instantdisclosure, which do not depart from the instant invention. Hence, thefollowing descriptions are intended to illustrate some particularembodiments of the invention, and not to exhaustively specify allpermutations, combinations and variations thereof.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. The terminology used in thedescription of the invention herein is for the purpose of describingparticular embodiments only and is not intended to be limiting of theinvention.

All publications, patent applications, patents and other referencescited herein are incorporated by reference in their entireties for theteachings relevant to the sentence and/or paragraph in which thereference is presented.

Unless the context indicates otherwise, it is specifically intended thatthe various features of the invention described herein May be used inany combination. Moreover, the present invention also contemplates thatin some embodiments of the invention, any feature or combination offeatures set forth herein may be excluded or omitted. To illustrate, ifthe specification states that a composition comprises components A, Band C, it is specifically intended that any of A, 1 or C, or acombination thereof, may be omitted and disclaimed singularly or in anycombination.

As used in the description of the invention and the appended claims, thesingular forms “a,” “an” and “the” are intended to include the pluralforms as well, unless the context clearly indicates otherwise.

Also as used herein, “and/or” refers to and encompasses any and allpossible combinations of one or more of the associated listed items, aswell as the lack of combinations when interpreted in the alternative(“or”).

The term “about,” as used herein when referring to a measurable valuesuch as a dosage or time period and the like refers to variations of±20%, ±10%, ±5%, ±1%, ±0.5%, or even ±0.1% of the specified amount.

As used herein, phrases such as “between X and Y” and “between about Xand Y” should be interpreted to include X and Y. As used herein, phrasessuch as “between about X and Y” mean “between about X and about Y” andphrases such as “from about X to Y” mean “from about X to about Y.”

The term “comprise,” “comprises” and “comprising” as used herein,specify the presence of the stated features, integers, steps,operations, elements, and/or components, but do not preclude thepresence or addition of one or more other features, integers, steps,operations, elements, components, and/or groups thereof.

As used herein, the transitional phrase “consisting essentially of”means that the scope of a claim is to be interpreted to encompass thespecified materials or steps recited in the claim and those that do notmaterially affect the basic and novel characteristic(s) of the claimedinvention. Thus, the term “consisting essentially of” when used in aclaim of this invention is not intended to be interpreted to beequivalent to “comprising.”

As used herein, “chimeric” refers to a nucleic acid molecule or apolypeptide in which at least two components are derived from differentsources (e.g., different organisms, different coding regions).

“Complement” as used herein can mean 100% complementarity with thecomparator nucleotide sequence or it can mean less than 100%complementarity (e.g., about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%,78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and the like, complementarity).

The terms “complementary” or “complementarity,” as used herein, refer tothe natural binding of polynucleotides under permissive salt andtemperature conditions by base-pairing. For example, the sequence“A-G-T” binds to the complementary sequence “T-C-A.” Complementaritybetween two single-stranded molecules may be “partial,” in which onlysome of the nucleotides bind, or it may be complete when totalcomplementarity exists between the single stranded molecules. The degreeof complementarity between nucleic acid strands has significant effectson the efficiency and strength of hybridization between nucleic acidstrands.

As used herein, “contact”, contacting”, “contacted,” and grammaticalvariations thereof, refer to placing the components of a desiredreaction together under conditions suitable for carrying out the desiredreaction (e.g., integration, transformation, site-specific cleavage(nicking, cleaving), amplifying, site specific targeting of apolypeptide of interest and the like). The methods and conditions forcarrying out such reactions are well known in the art (See, e.g.,Gasiunas et al. (2012) Proc. Natl. Acad. Sci. 109:E2579-E2586; M. R.Green and J. Sambrook (2012) Molecular Cloning: A Laboratory Manual. 4thEd., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.).

To “control” an organism (e.g., insect pest) means to inhibit, through atoxic effect, the ability of an organism (e.g., insect pest) to survive,grow, feed, and/or reproduce, or to limit damage or loss in crop plantsthat is related to the activity of the organism. To “control” anorganism may or may not mean killing the organism, although itpreferably means killing the organism.

“Pesticidally effective amount,” “effective pest controlling amount,” or“effective insect-controlling amount” means that concentration or amountof a polypeptide that inhibits, through a toxic effect, the ability ofpests or insects, respectively, to survive, grow, feed and/or reproduce,or to limit pest- or insect-related damage or loss in crop plants.“Pesticidally effective amount,” “effective pest controlling amount,” or“effective insect-controlling amount” may or may not mean killing thepests or insects, respectively, although it preferably means killing thepests or insects.

As used herein “pesticidal,” or “insecticidal,” and the like, refer tothe ability of a modified Vip3 polypeptide to control a pest organism oran amount of a modified Vip3 polypeptide that may control a pestorganism as defined herein. Thus, a pesticidal modified Vip3 polypeptidemay kill or inhibit the ability of a pest organism (e.g., insect pest)to survive, grow, feed, and/or reproduce. In some embodiments, amodified Vip3 polypeptide of the invention may be pesticidal orinsecticidal.

A “fragment” or “portion” of a nucleotide sequence or an amino acidsequence of the invention will be understood to mean a nucleotide or anamino acid sequence of reduced length relative (e.g., reduced by 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or morenucleotides) to a reference nucleic acid, nucleotide sequence, or anamino acid sequence and comprising, consisting essentially of and/orconsisting of a nucleotide sequence of contiguous nucleotides identicalor almost identical (e.g., 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%,79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99% identical) to the reference nucleicacid, nucleotide sequence, or amino acid sequence. Such a fragment orportion according to the invention may be, where appropriate, includedin a larger polynucleotide or polypeptide of which it is a constituent.

Thus, for example, fragments of Vip3 polypeptides may be used to carryout some embodiments of the present invention. In some embodiments, theterm “Vip3 polypeptide” refers to full length as well as portions orfragments of Vip3 polypeptides. In general, such fragments are at least20 or 30 contiguous amino acid residues in length. In some embodiments,a fragment of a Vip3 polypeptide may be at least about 20, 30, 40, 50,60, 70, 80, 90, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350,375, 400, 425, 450, 475, 500, 525, 550, 575 or more contiguous aminoacid residues in length. In some embodiments, a fragment of a Vip3polypeptide may be less than about 20, 30, 40, 50, 60, 70, 80, 90, 100,125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450,475, 500, 525, 550, 575 or more contiguous amino acid residues inlength. The fragment may comprise at least one, two, three or four ofVip3 polypeptide Domain I, Domain II, Domain III, and/or Domain IV,optionally with 1, 2, 3, 5, 7, 10, 12, 20, 30, 40, 50, 100 or moreadditional contiguous N-terminal and/or C-terminal amino acid residues.A Vip3 fragment may comprise all or a portion of Domain I, II, III, orIV. The length of the fragment (i.e., the number of contiguous aminoacid residues) may be about 10%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%,60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more of the full-length Vip3polypeptide. The fragment may comprise, consist essentially of and/orconsist of an amino acid sequence of contiguous amino acids identical oralmost identical (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%identical) to a corresponding portion of the Vip3 polypeptide. In someembodiments, the modified Vip3 polypeptide may comprise at least twofragments of a Vip3 polypeptide. In some embodiments, the modified Vip3polypeptide may comprise a first fragment of a Vip3 polypeptide and asecond fragment, wherein said first fragment may comprise about 100amino acid residues to about 600 amino acid residues, and any value orrange therein, and said second fragment may comprise about 100 to about300 amino acid residues, and any value or range therein.

In some embodiments, fragments of carbohydrate binding modules (CBMs)may be used to carry out some embodiments of the present invention. Insome embodiments, the term “carbohydrate binding module” (CBM) refers tofull length CBMs as well as portions or fragments of CBMs. In general,such fragments are at least 50 contiguous amino acid residues in length.In some embodiments, a fragment of a CBM may be at least about 50, 60,70, 80, 90, 100, 125, 150, 175, 200, 225, 250, 275, 300, or morecontiguous amino acid residues in length. In some embodiments, afragment of a CBM polypeptide may be less than about 50, 60, 70, 80, 90,100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425,450, 475, 500, 525, 550, 575 or more contiguous amino acid residues inlength. The fragment may optionally comprise 1, 2, 3, 5, 7, 10, 12, 20,30, 40, 50, 100 or more additional contiguous N-terminal and/orC-terminal amino acid residues. The length of the fragment (i.e., thenumber of contiguous amino acid residues) may be about 10%, 20%, 25%,30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% ormore of the full-length CBM. The fragment may comprise, consistessentially of and/or consist of an amino acid sequence of contiguousamino acids identical or almost identical (e.g., 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% identical) to a CBM from a carbohydrateactive enzyme.

Optionally, the CBM fragment comprises the metal binding domain (and/orany other known functional domain).

In some embodiments, the CBM fragment may comprise, consist essentiallyof, or consist of a CBM having an N and/or C terminal truncation. Insome embodiments, the CBM may comprise a C-terminal truncation of about1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 26, 27, 28, 29, 30 amino acid residues, and thelike, or any range or variable therein. In some embodiments, the CBM maycomprise an N-terminal truncation of about 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28, 29, 30 amino acid residues, and the like, or any range or variabletherein. In some embodiments, a CBM useful with this invention can betruncation at both its N-terminal end and C-terminal end.

As used herein, the term “gene” refers to a nucleic acid moleculecapable of being used to produce mRNA, antisense RNA, miRNA,anti-microRNA antisense oligodeoxyribonucleotide (AMO) and the like.Genes may or may not be capable of being used to produce a functionalprotein or gene product. Genes may include both coding and non-codingregions (e.g., introns, regulatory elements, promoters, enhancers,termination sequences and/or 5′ and 3′ untranslated regions). A gene maybe “isolated” by which is meant a nucleic acid that is substantially oressentially free from components normally found in association with thenucleic acid in its natural state. Such components include othercellular material, culture medium from recombinant production, and/orvarious chemicals used in chemically synthesizing the nucleic acid.

A “heterologous” or a “recombinant” nucleotide sequence is a nucleotidesequence not naturally associated with a host cell into which it isintroduced, including non-naturally occurring multiple copies of anaturally occurring nucleotide sequence and nucleotide sequences thatare introduced into a host cell in a form that is not naturallyoccurring (e.g., operably linked to regulatory sequence(s) that do notnaturally occur with that nucleotide sequence).

Different nucleic acids or proteins having homology are referred toherein as “homologues.” The term homologue includes homologous sequencesfrom the same and different species and orthologous sequences from thesame and different species. “Homology” refers to the level of similarityor identity between two or more nucleic acid and/or amino acid sequencesin terms of percent of positional identity (i.e., sequence similarity oridentity). Homology also refers to the concept of similar functionalproperties among different nucleic acids or proteins. Thus, thecompositions and methods of the invention may comprise homologues to thenucleotide sequences and polypeptide sequences of this invention.“Orthologous,” as used herein, refers to homologous nucleotide sequencesand/or amino acid sequences in different species that arose from acommon ancestral gene during speciation. A homologue of a nucleotidesequence of this invention has a substantial sequence identity (e.g., atleast about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%,82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, and/or 100%) to said nucleotide sequence of theinvention. Thus, for example, a homologue of a CBM useful with thisinvention may be at least about 70% homologous or more to any one of theCBM sequences provided herein, wherein the CBM sequence homologue hasthe function of binding carbohydrate(s). In some embodiments, the CBMsequence homologue has the function of binding cellulose and/orglucomannan. In some embodiments, the CBM sequence homologue has thefunction of binding internally on glycan chains. In some embodiments, ahomologue of a Vip3 polypeptide useful with this invention may be about70% homologous or more to any one of the Vip3 polypeptide sequencesprovided herein, wherein the homologue has pesticidal activity.

Thus, a homologue of a Vip3 polypeptide includes, but is not limited to:(1) polypeptides which are at least about 70% to at least about 90%(e.g., 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%,83%, 84%, 85%, 86%, 87%, 88%, 89%, 90% 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, 100%) identical at the sequence level to a member of theVip3 class of polypeptides while also retaining pesticidal activity; (2)polypeptides which are cross-reactive to antibodies whichimmunologically recognize a member of the Vip3 class of polypeptides,(3) polypeptides which are cross-reactive with a receptor to a member ofthe Vip3 class of polypeptides and retain pesticidal activity, and (4)polypeptides, which are at least about 70% to at least about 90% (e.g.,70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 90% 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, 100%) identical at the sequence level to the toxic core regionof a member of the Vip3 class of polypeptides, while also retainingpesticidal activity. Vip3 homologues have been disclosed in WO 98/18932,WO 98/33991, WO 98/00546, and WO 99/57282. An alignment of Vip3A andVip3D polypeptides is provided in FIG. 1 and shows the substantialsimilarity in secondary structure.

Thus, in some embodiments of the invention, the polypeptides are atleast 70% identical at the sequence level to a member of the Vip3 classof polypeptides and/or to the toxic core region of a member of the Vip3class of polypeptides, while also retaining pesticidal activity. In someembodiments of the invention, the polypeptides are at least 80%identical at the sequence level to a member of the Vip3 class ofpolypeptides and/or to the toxic core region of a member of the Vip3class of polypeptides, while also retaining pesticidal activity. In someembodiments, the polypeptides are at least 90% identical at the sequencelevel to a member of the Vip3 class of polypeptides and/or to the toxiccore region of a member of the Vip3 class of polypeptides, while alsoretaining pesticidal activity.

As used herein, hybridization, hybridize, hybridizing, and grammaticalvariations thereof, refer to the binding of two fully complementarynucleotide sequences or substantially complementary sequences in whichsome mismatched base pairs are present. The conditions for hybridizationare well known in the art and vary based on the length of the nucleotidesequences and the degree of complementarity between the nucleotidesequences. In some embodiments, the conditions of hybridization can behigh stringency, or they can be medium stringency or low stringencydepending on the amount of complementarity and the length of thesequences to be hybridized. The conditions that constitute low, mediumand high stringency for purposes of hybridization between nucleotidesequences are well known in the art (See, e.g., Gasiunas et al. (2012)Proc. Natl. Acad. Sci. 109:E2579-E2586; M. R. Green and J. Sambrook(2012) Molecular Cloning: A Laboratory Manual. 4th Ed., Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y.).

As used herein, the terms “increase,” “increasing,” “increased,”“enhance,” “enhanced,” “enhancing,” and “enhancement” (and grammaticalvariations thereof) describe an elevation of at least about 25%, 50%,75%, 100%, 150%, 200%, 300%, 400%, 500% or more as compared to acontrol.

A “native,” or “wild type” nucleic acid, nucleotide sequence,polypeptide or amino acid sequence refers to a naturally occurring orendogenous nucleic acid, nucleotide sequence, polypeptide or amino acidsequence. Thus, for example, a “wild type Vip3” is a Vip3 that isnaturally occurring in or endogenous to the organism.

In contrast, a “heterologous” nucleic acid or polypeptide is a nucleicacid or polypeptide that is not naturally associated with a host cellinto which it is introduced or is introduced in a form that is notnaturally found in the cell into which it is being introduced.

Also as used herein, the terms “nucleic acid,” “nucleic acid molecule,”“nucleic acid construct,” “nucleotide sequence” and “polynucleotide”refer to RNA or DNA that is linear or branched, single or doublestranded, or a hybrid thereof. The term also encompasses RNA/DNAhybrids. When dsRNA is produced synthetically, less common bases, suchas inosine, 5-methylcytosine, 6-methyladenine, hypoxanthine and otherscan also be used for antisense, dsRNA, and ribozyme pairing. Forexample, polynucleotides that contain C-5 propyne analogues of uridineand cytidine have been shown to bind RNA with high affinity and to bepotent antisense inhibitors of gene expression. Other modifications,such as modification to the phosphodiester backbone, or the 2′-hydroxyin the ribose sugar group of the RNA can also be made. The nucleic acidconstructs of the present disclosure can be DNA or RNA, but arepreferably DNA. Thus, although the nucleic acid constructs of thisinvention may be described and used in the form of DNA, depending on theintended use, they may also be described and used in the form of RNA.

A “synthetic” nucleic acid or nucleotide sequence, as used herein,refers to a nucleic acid or nucleotide sequence that is not found innature but is constructed by the hand of man and as a consequence is nota product of nature.

As used herein, the term “nucleotide sequence” refers to a heteropolymerof nucleotides or the sequence of these nucleotides from the 5′ to 3′end of a nucleic acid molecule and includes DNA or RNA molecules,including cDNA, a DNA fragment or portion, genomic DNA, synthetic (e.g.,chemically synthesized) DNA, plasmid DNA, mRNA, and anti-sense RNA, anyof which can be single stranded or double stranded. The terms“nucleotide sequence” “nucleic acid,” “nucleic acid molecule,”“oligonucleotide” and “polynucleotide” are also used interchangeablyherein to refer to a heteropolymer of nucleotides. Except as otherwiseindicated, nucleic acid molecules and/or nucleotide sequences providedherein are presented herein in the 5′ to 3′ direction, from left toright and are represented using the standard code for representing thenucleotide characters as set forth in the U.S. sequence rules, 37 CFR §§1.821-1.825 and the World Intellectual Property Organization (WIPO)Standard ST.25. A “5′ region” as used herein can mean the region of apolynucleotide that is nearest the 5′ end. Thus, for example, an elementin the 5′ region of a polynucleotide can be located anywhere from thefirst nucleotide located at the 5′ end of the polynucleotide to thenucleotide located halfway through the polynucleotide. A “3′ region” asused herein can mean the region of a polynucleotide that is nearest the3′ end. Thus, for example, an element in the 3′ region of apolynucleotide can be located anywhere from the first nucleotide locatedat the 3′ end of the polynucleotide to the nucleotide located halfwaythrough the polynucleotide.

As used herein, the term “percent sequence identity” or “percentidentity” refers to the percentage of identical nucleotides in a linearpolynucleotide sequence of a reference (“query”) polynucleotide molecule(or its complementary strand) as compared to a test (“subject”)polynucleotide molecule (or its complementary strand) when the twosequences are optimally aligned. In some embodiments, “percent identity”can refer to the percentage of identical amino acids in an amino acidsequence.

The terms “modify,” “modifying” and/or “modification” (and grammaticalvariants thereof) as used herein with regard to Vip3 polypeptides andthe polynucleotides encoding the Vip3 polypeptides refers to changingthe wild-type or reference Vip3 polypeptide and its correspondingnucleotide sequence to include a heterologous carbohydrate bindingmodule. In some embodiments, the toxicity of the modified Vip3polypeptide produced is changed relative to the toxicity of the wildtype or reference Vip3 polypeptide. A “change in toxicity” includes, butis not limited to, an increase and/or decrease in toxicity as itpertains to any particular target organism and/or a change in theorganism(s) that are targeted (e.g., that the modified Vip3 polypeptideis now pesticidal against one or more additional organism(s) as comparedto the wild-type or reference Vip3 polypeptide), including a change inmaximum activity, a change in LC₅₀, and/or a change in the time toachieve toxicity.

As used herein, the terms “reduce,” “reduced,” “reducing,” “reduction,”“diminish,” “suppress,” and “decrease” (and grammatical variationsthereof), describe, for example, a decrease of at least about 5%, 10%,15%, 20%, 25%, 35%, 50%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100%and the like, or any value or range therein, as compared to a control.In some embodiments, a reduction may result in no or essentially no(i.e., an insignificant amount, e.g., less than about 10% or even 5%)detectable activity or amount. Thus, in some embodiments, a transgenicplant comprising a nucleic acid molecule that expresses the modifiedVip3 polypeptide may reduce damage caused by a plant pest by at leastabout 5%, 10%, 15%, 20%, 25%, 35%, 50%, 75%, 80%, 85%, 90%, 95%, 97%,98%, 99%, or 100%, and the like, or any value or range therein, ascompared to a control (e.g., a plant that does not comprise said nucleicacid molecule that expresses the modified Vip3 polypeptide).

As used herein, “regulatory sequence(s)” means nucleotide sequence(s)located upstream (5′ non-coding sequences), within or downstream (3′non-coding sequences) of a coding sequence, which influence thetranscription, RNA processing or stability, or translation of theassociated coding sequence. Regulatory sequences include, but are notlimited to, promoters, enhancers, exons, introns, translation leadersequences, termination signals, and polyadenylation signal sequences.Regulatory sequences include natural and synthetic sequences as well assequences that can be a combination of synthetic and natural sequences.An “enhancer” is a nucleotide sequence that can stimulate promoteractivity and can be an innate element of the promoter or a heterologouselement inserted to enhance the level or tissue specificity of apromoter. The primary sequence can be present on either strand of adouble-stranded DNA molecule, and is capable of functioning even whenplaced either upstream or downstream from the promoter. The meaning ofthe term “promoter” can include “promoter regulatory sequences.”

As used herein “sequence identity” refers to the extent to which twooptimally aligned polynucleotide or peptide sequences are invariantthroughout a window of alignment of components, e.g., nucleotides oramino acids. “Identity” can be readily calculated by known methodsincluding, but not limited to, those described in: ComputationalMolecular Biology (Lesk, A. M., ed.) Oxford University Press, New York(1988); Biocomputing: Informatics and Genome Projects (Smith, D. W.,ed.) Academic Press, New York (1993); Computer Analysis of SequenceData, Part I (Griffin, A. M., and Griffin, H. G., eds.) Humana Press,New Jersey (1994); Sequence Analysis in Molecular Biology (von Heinje,G., ed.) Academic Press (1987); and Sequence Analysis Primer (Gribskov,M. and Devereux, J., eds.) Stockton Press, New York (1991).

As used herein, the phrase “substantially identical,” or “substantialidentity” in the context of two nucleic acid molecules, nucleotidesequences or protein sequences, refers to two or more sequences orsubsequences that have at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%,77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and/or 100% nucleotide oramino acid residue identity, when compared and aligned for maximumcorrespondence, as measured using one of the following sequencecomparison algorithms or by visual inspection. In some embodiments ofthe invention, the substantial identity exists over a region of thesequences that is at least about 50 residues to about 750 residues inlength. Thus, in some embodiments, substantial identity exists over aregion of the sequences that is at least about 50 residues to about 250residues in length, about 75 residues to about 225 residues in length,about 100 residues to about 200 residues in length, about 125 residuesto about 175 residues in length, about 200 residues to about 400residues in length, about 300 residues to about 450 residues in length,about 400 residues to about 500 residues in length, about 500 residuesto about 550 residues in length, about 550 residues to about 650residues in length, and/or about 650 residues to about 750 residues inlength, or any value or range therein.

For sequence comparison, typically one sequence acts as a referencesequence to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are entered into acomputer, subsequence coordinates are designated if necessary, andsequence algorithm program parameters are designated. The sequencecomparison algorithm then calculates the percent sequence identity forthe test sequence(s) relative to the reference sequence, based on thedesignated program parameters.

Optimal alignment of sequences for aligning a comparison window are wellknown to those skilled in the art and may be conducted by tools such asthe local homology algorithm of Smith and Waterman, the homologyalignment algorithm of Needleman and Wunsch, the search for similaritymethod of Pearson and Lipman, and optionally by computerizedimplementations of these algorithms such as GAP, BESTFIT, FASTA, andTFASTA available as part of the GCG® Wisconsin Package® (Accelrys Inc.,San Diego, Calif.). An “identity fraction” for aligned segments of atest sequence and a reference sequence is the number of identicalcomponents which are shared by the two aligned sequences divided by thetotal number of components in the reference sequence segment, i.e., theentire reference sequence or a smaller defined part of the referencesequence. Percent sequence identity is represented as the identityfraction multiplied by 100. The comparison of one or more polynucleotidesequences may be to a full-length polynucleotide sequence or a portionthereof, or to a longer polynucleotide sequence. For purposes of thisinvention “percent identity” may also be determined using BLASTX version2.0 for translated nucleotide sequences and BLASTN version 2.0 forpolynucleotide sequences.

Software for performing BLAST analyses is publicly available through theNational Center for Biotechnology Information. This algorithm involvesfirst identifying high scoring sequence pairs (HSPs) by identifyingshort words of length W in the query sequence, which either match orsatisfy some positive-valued threshold score T when aligned with a wordof the same length in a database sequence. T is referred to as theneighborhood word score threshold (Altschul et al., 1990). These initialneighborhood word hits act as seeds for initiating searches to findlonger HSPs containing them. The word hits are then extended in bothdirections along each sequence for as far as the cumulative alignmentscore can be increased. Cumulative scores are calculated using, fornucleotide sequences, the parameters M (reward score for a pair ofmatching residues; always >0) and N (penalty score for mismatchingresidues; always <0). For amino acid sequences, a scoring matrix is usedto calculate the cumulative score. Extension of the word hits in eachdirection are halted when the cumulative alignment score falls off bythe quantity X from its maximum achieved value, the cumulative scoregoes to zero or below due to the accumulation of one or morenegative-scoring residue alignments, or the end of either sequence isreached. The BLAST algorithm parameters W, T, and X determine thesensitivity and speed of the alignment. The BLASTN program (fornucleotide sequences) uses as defaults a wordlength (W) of 11, anexpectation (E) of 10, a cutoff of 100, M=5, N=−4, and a comparison ofboth strands. For amino acid sequences, the BLASTP program uses asdefaults a wordlength (W) of 3, an expectation (E) of 10, and theBLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci.USA 89: 10915 (1989)).

In addition to calculating percent sequence identity, the BLASTalgorithm also performs a statistical analysis of the similarity oridentity between two sequences (see, e.g., Karlin & Altschul, Proc.Nat'l. Acad. Sci. USA 90: 5873-5787 (1993)). One measure of similarityor identity provided by the BLAST algorithm is the smallest sumprobability (P(N)), which provides an indication of the probability bywhich a match between two nucleotide or amino acid sequences would occurby chance. For example, a test nucleic acid sequence is consideredsimilar to a reference sequence if the smallest sum probability in acomparison of the test nucleotide sequence to the reference nucleotidesequence is less than about 0.1 to less than about 0.001. Thus, in someembodiments of the invention, the smallest sum probability in acomparison of the test nucleotide sequence to the reference nucleotidesequence is less than about 9.001.

Two nucleotide sequences may also be considered to be substantiallycomplementary when the two sequences hybridize to each other understringent conditions. In some embodiments, two nucleotide sequencesconsidered to be substantially complementary hybridize to each otherunder highly stringent conditions.

“Stringent hybridization conditions” and “stringent hybridization washconditions” in the context of nucleic acid hybridization experimentssuch as Southern and Northern hybridizations are sequence dependent, andare different under different environmental parameters. An extensiveguide to the hybridization of nucleic acids is found in TijssenLaboratory Techniques in Biochemistry and MolecularBiology-Hybridization with Nucleic Acid Probes part I chapter 2“Overview of principles of hybridization and the strategy of nucleicacid probe assays” Elsevier, New York (1993). Generally, highlystringent hybridization and wash conditions are selected to be about 5°C. lower than the thermal melting point (T_(m)) for the specificsequence at a defined ionic strength and pH.

The T_(m) is the temperature (under defined ionic strength and pH) atwhich 50% of the target sequence hybridizes to a perfectly matchedprobe. Very stringent conditions are selected to be equal to the T_(m)for a particular probe. An example of stringent hybridization conditionsfor hybridization of complementary nucleotide sequences which have morethan 100 complementary residues on a filter in a Southern or northernblot is 50% formamide with 1 mg of heparin at 42° C., with thehybridization being carried out overnight. An example of highlystringent wash conditions is 0.1 5M NaCl at 72° C. for about 15 minutes.An example of stringent wash conditions is a 0.2×SSC wash at 65° C. for15 minutes (see, Sambrook, infra, for a description of SSC buffer).Often, a high stringency wash is preceded by a low stringency wash toremove background probe signal. An example of a medium stringency washfor a duplex of, e.g., more than 100 nucleotides, is 1×SSC at 45° C. for15 minutes. An example of a low stringency wash for a duplex of, e.g.,more than 100 nucleotides, is 4-6×SSC at 40° C. for 15 minutes. Forshort probes (e.g., about 10 to 50 nucleotides), stringent conditionstypically involve salt concentrations of less than about 1.0 M Na ion,typically about 0.01 to 1.0 M Na ion concentration (or other salts) atpH 7.0 to 8.3, and the temperature is typically at least about 30° C.Stringent conditions can also be achieved with the addition ofdestabilizing agents such as formamide. In general, a signal to noiseratio of 2× (or higher) than that observed for an unrelated probe in theparticular hybridization assay indicates detection of a specifichybridization. Nucleotide sequences that do not hybridize to each otherunder stringent conditions are still substantially identical if theproteins that they encode are substantially identical. This can occur,for example, when a copy of a nucleotide sequence is created using themaximum codon degeneracy permitted by the genetic code.

The following are examples of sets of hybridization/wash conditions thatmay be used to clone homologous nucleotide sequences that aresubstantially identical to reference nucleotide sequences of theinvention. In one embodiment, a reference nucleotide sequence hybridizesto the “test” nucleotide sequence in 7% sodium dodecyl sulfate (SDS),0.5 M NaPO₄, 1 mM EDTA at 50° C. with washing in 2×SSC, 0.1% SDS at 50°C. In some embodiments, the reference nucleotide sequence hybridizes tothe “test” nucleotide sequence in 7% sodium dodecyl sulfate (SDS), 0.5 MNaPO₄, 1 mM EDTA at 50° C. with washing in 1×SSC, 0.1% SDS at 50° C. orin 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO₄, 1 mM EDTA at 50° C.with washing in 0.5×SSC, 0.1% SDS at 50° C. In some embodiments, thereference nucleotide sequence hybridizes to the “test” nucleotidesequence in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO₄, 1 mM EDTA at50° C. with washing in 0.1×SSC, 0.1% SDS at 50° C., or in 7% sodiumdodecyl sulfate (SDS), 0.5 M NaPO₄, 1 mM EDTA at 50° C. with washing in0.1×SSC, 0.1% SDS at 65° C.

Any nucleotide sequence and/or recombinant nucleic acid molecule of thisinvention may be codon optimized for expression in any species ofinterest. Codon optimization is well known in the art and involvesmodification of a nucleotide sequence for codon usage bias using speciesspecific codon usage tables. The codon usage tables are generated basedon a sequence analysis of the most highly expressed genes for thespecies of interest. When the nucleotide sequences are to be expressedin the nucleus, the codon usage tables are generated based on a sequenceanalysis of highly expressed nuclear genes for the species of interest.The modifications of the nucleotide sequences are determined bycomparing the species specific codon usage table with the codons presentin the native polynucleotide sequences. As is understood in the art,codon optimization of a nucleotide sequence results in a nucleotidesequence having less than 100% identity (e.g., 70%, 71%, 72%, 73%, 74%,75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and the like) tothe native nucleotide sequence but which still encodes a polypeptidehaving the same function as that encoded by the original, nativenucleotide sequence. Thus, in some embodiments of the invention, thenucleotide sequence and/or recombinant nucleic acid molecule of thisinvention may be codon optimized for expression in the particularspecies of interest (e.g., a plant such as corn, soybean, sugar cane,sugar beet, rice or wheat).

In some embodiments, the recombinant nucleic acid molecules, nucleotidesequences and polypeptides of the invention are “isolated.” An“isolated” nucleic acid molecule, an “isolated” nucleotide sequence oran “isolated” polypeptide is a nucleic acid molecule, nucleotidesequence or polypeptide that, by the hand of man, exists apart from itsnative environment and is therefore not a product of nature. An isolatednucleic acid molecule, nucleotide sequence or polypeptide may exist in apurified form that is at least partially separated from at least some ofthe other components of the naturally occurring organism or virus, forexample, the cell or viral structural components or other polypeptidesor nucleic acids commonly found associated with the polynucleotide. Insome embodiments, the isolated nucleic acid molecule, the isolatednucleotide sequence and/or the isolated polypeptide is at least about1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more pure.

In some embodiments, an isolated nucleic acid molecule, nucleotidesequence or polypeptide may exist in a non-native environment such as,for example, a recombinant host cell. Thus, for example, with respect tonucleotide sequences, the term “isolated” means that it is separatedfrom the chromosome and/or cell in which it naturally occurs. Apolynucleotide is also isolated if it is separated from the chromosomeand/or cell in which it naturally occurs in and is then inserted into agenetic context, a chromosome and/or a cell in which it does notnaturally occur (e.g., a different host cell, different regulatorysequences, and/or different position in the genome than as found innature). Accordingly, the recombinant nucleic acid molecules, nucleotidesequences and their encoded polypeptides are “isolated” in that, by thehand of man, they exist apart from their native environment andtherefore are not products of nature, however, in some embodiments, theycan be introduced into and exist in a recombinant host cell.

In any of the embodiments described herein, the nucleotide sequencesand/or recombinant nucleic acid molecules of the invention can beoperatively associated with a variety of promoters and other regulatoryelements for expression in cells of various organisms. Thus, in someembodiments, a recombinant nucleic acid of this invention may furthercomprise one or more promoters operably linked to one or more nucleotidesequences.

By “operably linked” or “operably associated” as used herein, it ismeant that the indicated elements are functionally related to eachother, and are also generally physically related. Thus, the term“operably linked” or “operably associated” as used herein, refers tonucleotide sequences on a single nucleic acid molecule that arefunctionally associated. Thus, a first nucleotide sequence that isoperably linked to a second nucleotide sequence means a situation whenthe first nucleotide sequence is placed in a functional relationshipwith the second nucleotide sequence. For instance, a promoter isoperably associated with a nucleotide sequence if the promoter effectsthe transcription or expression of said nucleotide sequence. Thoseskilled in the art will appreciate that the control sequences (e.g.,promoter) need not be contiguous with the nucleotide sequence to whichit is operably associated, as long as the control sequences function todirect the expression thereof. Thus, for example, interveninguntranslated, yet transcribed, sequences may be present between apromoter and a nucleotide sequence, and the promoter may still beconsidered “operably linked” to the nucleotide sequence.

A “promoter” is a nucleotide sequence that controls or regulates thetranscription of a nucleotide sequence (i.e., a coding sequence) that isoperably associated with the promoter. Typically, a “promoter” refers toa nucleotide sequence that contains a binding site for RNA polymerase IIand directs the initiation of transcription. In general, promoters arefound 5′, or upstream, relative to the start of the coding region of thecorresponding coding sequence. The promoter region may comprise otherelements that act as regulators of gene expression. These include a TATAbox consensus sequence, and often a CAAT box consensus sequence(Breathnach and Chambon, (1981) Annu. Rev. Biochem. 50:349). In plants,the CAAT box may be substituted by the AGGA box (Messing et al., (1983)in Genetic Engineering of Plants, T. Kosuge, C. Meredith and A.Hollaender (eds.), Plenum Press, pp. 211-227).

Any promoter useful for initiation of transcription in a cell of a plantor bacteria may be used in the expression cassettes of the presentinvention. Promoters may include, for example, constitutive, inducible,temporally regulated, developmentally regulated, chemically regulated,tissue-preferred and/or tissue-specific promoters for use in thepreparation of recombinant nucleic acid molecules, i.e., “chimericgenes” or “chimeric polynucleotides.” These various types of promotersare known in the art.

The choice of promoter will vary depending on the temporal and spatialrequirements for expression, and also depending on the host cell (e.g.,plant or bacteria) to be transformed. Promoters for many differentorganisms are well known in the art. Based on the extensive knowledgepresent in the art, the appropriate promoter can be selected for theparticular host organism of interest. Thus, for example, much is knownabout promoters upstream of highly constitutively expressed genes inmodel organisms and such knowledge can be readily accessed andimplemented in other systems as appropriate.

Further, for example, expression of a heterologous polynucleotideencoding a modified Vip3 polypeptide of the invention may be in anyplant, plant part, (e.g., in leaves, in stalks or stems, in ears, ininflorescences (e.g. spikes, panicles, cobs, etc.), in roots, seedsand/or seedlings, and the like), plant cells, or bacterial cells. Forexample, in the case of a multicellular organism such as a plant whereexpression in a specific tissue or organ is desired, a tissue-specificor tissue preferred promoter may be used (e.g., a rootspecific/preferred promoter). In contrast, where expression in responseto a stimulus is desired a promoter inducible by stimuli or chemicalsmay be used. In some embodiments, protection against more than one typeof insect pest may be sought, and thus expression in multiple tissues isdesirable. Where continuous expression at a relatively constant level isdesired throughout the cells or tissues of an organism a constitutivepromoter may be chosen.

Thus, promoters useful with the invention include, but are not limitedto, those that drive expression of a nucleotide sequence constitutively,those that drive expression when induced, and those that driveexpression in a tissue- or developmentally-specific or -preferredmanner. These various types of promoters are known in the art. Promoterscan be identified in and isolated from the plant, yeast, or bacteria tobe transformed and then inserted into the expression cassette to be usedin transformation of the plant, yeast, or bacteria.

Non-limiting examples of a promoter include the promoter of the RubisCosmall subunit gene 1 (PrbcS1), the promoter of the actin gene (Pactin),the promoter of the nitrate reductase gene (Pnr) and the promoter ofduplicated carbonic anhydrase gene 1 (Pdca1) (See, Walker et al. PlantCell Rep. 23:727-735 (2005); Li et al. Gene 403:132-142 (2007); Li etal. Mol Biol. Rep. 37:1143-1154 (2010)). PrbcS1 and Pactin areconstitutive promoters and Pnr and Pdca1 are inducible promoters. Pnr isinduced by nitrate and repressed by ammonium (Li et al. Gene 403:132-142(2007)) and Pdca1 is induced by salt (Li et al. Mol Biol. Rep.37:1143-1154 (2010)).

Examples of constitutive promoters useful for plants include, but arenot limited to, cestrum virus promoter (cmp) (U.S. Pat. No. 7,166,770),the rice actin 1 promoter (Wang et al. (1992) Mol. Cell. Biol.12:3399-3406; as well as U.S. Pat. No. 5,641,876), CaMV 35S promoter(Odell et al. (1985) Nature 313:810-812), CaMV 19S promoter (Lawton etal. (1987) Plant Mol. Biol. 9:315-324), nos promoter (Ebert et al.(1987) Proc. Natl. Acad. Sci USA 84:5745-5749), Adh promoter (Walker etal. (1987) Proc. Natl. Acad. Sci. USA 84:6624-6629), sucrose synthasepromoter (Yang & Russell (1990) Proc. Natl. Acad. Sci. USA87:4144-4148), and the ubiquitin promoter. The constitutive promoterderived from ubiquitin accumulates in many cell types. Ubiquitinpromoters have been cloned from several plant species for use intransgenic plants, for example, sunflower (Binet et al., 1991. PlantScience 79: 87-94), maize (Christensen et al., 1989. Plant Molec. Biol.12: 619-632), and arabidopsis (Norris et al. 1993. Plant Molec. Biol.21:895-906). The maize ubiquitin promoter (UbiP) has been developed intransgenic monocot systems and its sequence and vectors constructed formonocot transformation are disclosed in the patent publication EP 0 342926. The ubiquitin promoter is suitable for the expression of thenucleotide sequences of the invention in transgenic plants, especiallymonocotyledons. Further, the promoter expression cassettes described byMcElroy et al. (Mol. Gen. Genet. 231: 150-160 (1991)) can be easilymodified for the expression of the nucleotide sequences of the inventionand are particularly suitable for use in monocotyledonous hosts.

In some embodiments, tissue specific/tissue preferred promoters may beused for expression of a heterologous polynucleotide in a plant cell.Tissue specific or preferred expression patterns include, but are notlimited to, green tissue specific or preferred, root specific orpreferred, stem specific or preferred, and flower specific or preferred.Promoters suitable for expression in green tissue include many thatregulate genes involved in photosynthesis and many of these have beencloned from both monocotyledons and dicotyledons. In one embodiment, apromoter useful with the invention is the maize PEPC promoter from thephosphoenol carboxylase gene (Hudspeth & Grula, Plant Molec. Biol.12:579-589 (1989)). Non-limiting examples of tissue-specific promotersinclude those associated with genes encoding the seed storage proteins(such as β-conglycinin, cruciferin, napin and phaseolin), zein or oilbody proteins (such as oleosin), or proteins involved in fatty acidbiosynthesis (including acyl carrier protein, stearoyl-ACP desaturaseand fatty acid desaturases (fad 2-1)), and other nucleic acids expressedduring embryo development (such as Bce4, see, e.g., Kridl et al. (1991)Seed Sci. Res. 1:209-219; as well as EP Patent No. 255378).Tissue-specific or tissue-preferential promoters useful for theexpression of the nucleotide sequences of the invention in plants,particularly maize, include but are not limited to those that directexpression in root, pith, leaf or pollen. Such promoters are disclosed,for example, in WO 93/07278, herein incorporated by reference in itsentirety. Other non-limiting examples of tissue specific or tissuepreferred promoters useful with the invention the cotton rubiscopromoter disclosed in U.S. Pat. No. 6,040,504; the rice sucrose synthasepromoter disclosed in U.S. Pat. No. 5,604,121; the root specificpromoter described by de Framond (FEBS 290:103-106 (1991); EP 0 452 269to Ciba-Geigy); the stem specific promoter described in U.S. Pat. No.5,625,136 (to Ciba-Geigy) and which drives expression of the maize trpAgene; and the cestrum yellow leaf curling virus promoter disclosed in WO01/73087.

Additional examples of plant tissue-specific/tissue preferred promotersinclude, but are not limited to, the root hair-specific cis-elements(RHEs) (Kim et al. The Plant Cell 18:2958-2970 (2006)), theroot-specific promoters RCc3 (Jeong et al. Plant Physiol. 153:185-197(2010)) and RB7 (U.S. Pat. No. 5,459,252), the lectin promoter(Lindstrom et al. (1990) Der. Genet. 11:160-167; and Vodkin (1983) Prog.Clin. Biol. Res. 138:87-98), corn alcohol dehydrogenase 1 promoter(Dennis et al. (1984) Nucleic Acids Res. 12:3983-4000),S-adenosyl-L-methionine synthetase (SAMS) (Vander Mijnsbrugge et al.(1996) Plant and Cell Physiology, 37(8):1108-1115), corn lightharvesting complex promoter (Bansal et al. (1992) Proc. Natl. Acad. Sci.USA 89:3654-3658), corn heat shock protein promoter (O'Dell et al.(1985) EMBO J. 5:451-458; and Rochester et al. (1986) EMBO J.5:451-458), pea small subunit RuBP carboxylase promoter (Cashmore,“Nuclear genes encoding the small subunit of ribulose-1,5-bisphosphatecarboxylase” pp. 29-39 In: Genetic Engineering of Plants (Hollaendered., Plenum Press 1983; and Poulsen et al. (1986) Mol. Gen. Genet.205:193-200), Ti plasmid mannopine synthase promoter (Langridge et al.(1989) Proc. Natl. Acad. Sci. USA 86:3219-3223), Ti plasmid nopalinesynthase promoter (Langridge et al. (1989), supra), petunia chalconeisomerase promoter (van Tunen et al. (1988) EMBO J 7:1257-1263), beanglycine rich protein 1 promoter (Keller et al. (1989) Genes Dev.3:1639-1646), truncated CaMV 35S promoter (O'Dell et al. (1985) Nature313:810-812), potato patatin promoter (Wenzler et al. (1989) Plant Mol.Biol. 13:347-354), root cell promoter (Yamamoto et al. (1990) NucleicAcids Res. 18:7449), maize zein promoter (Kriz et al. (1987) Mol. Gen.Genet. 207:90-98; Langridge et al. (1983) Cell 34:1015-1022; Reina etal. (1990) Nucleic Acids Res. 18:6425; Reina et al. (1990) Nucleic AcidsRes. 18:7449; and Wandelt et al. (1989) Nucleic Acids Res. 17:2354),globulin-1 promoter (Belanger et al. (1991) Genetics 129:863-872),α-tubulin cab promoter (Sullivan et al. (1989) Mol. Gen. Genet.215:431-440), PEPCase promoter (Hudspeth & Grula (1989) Plant Mol. Biol.12:579-589), R gene complex-associated promoters (Chandler et al. (1989)Plant Cell 1:1175-1183), and chalcone synthase promoters (Franken et al.(1991) EMBO J. 10:2605-2612).

Particularly useful for seed-specific expression is the pea vicilinpromoter (Czako et al. (1992) Mol. Gen. Genet. 235:33-40; as well as theseed-specific promoters disclosed in U.S. Pat. No. 5,625,136. Usefulpromoters for expression in mature leaves are those that are switched atthe onset of senescence, such as the SAG promoter from Arabidopsis (Ganet al. (1995) Science 270:1986-1988).

In addition, promoters functional in chloroplasts may be used.Non-limiting examples of such promoters include the bacteriophage T3gene 9 5′ UTR and other promoters disclosed in U.S. Pat. No. 7,579,516.Other promoters useful with the invention include but are not limited tothe S-E9 small subunit RuBP carboxylase promoter and the Kunitz trypsininhibitor gene promoter (Kti3).

In some embodiments of the invention, inducible promoters may be used.Thus, for example, chemical-regulated promoters may be used to modulatethe expression of a gene in an organism through the application of anexogenous chemical regulator. Regulation of the expression of nucleotidesequences of the invention via promoters that are chemically regulatedenables the polypeptides of the invention to be synthesized only when,for example, a crop of plants are treated with the inducing chemicals.Depending upon the objective, the promoter may be a chemical-induciblepromoter, where application of a chemical induces gene expression, or achemical-repressible promoter, where application of the chemicalrepresses gene expression.

Chemical inducible promoters useful with plants are known in the art andinclude, but are not limited to, the maize In2-2 promoter, which isactivated by benzenesulfonamide herbicide safeners, the maize GSTpromoter, which is activated by hydrophobic electrophilic compounds thatare used as pre-emergent herbicides, and the tobacco PR-1a promoter,which is activated by salicylic acid (e.g., the PR1a system),steroid-responsive promoters (see, e.g., the glucocorticoid-induciblepromoter in Schena et al. (1991) Proc. Natl. Acad. Sci. USA 88,10421-10425 and McNellis et al. (1998) Plant J. 14, 247-257) andtetracycline-inducible and tetracycline-repressible promoters (see,e.g., Gatz et al. (1991) Mol. Gen. Genet. 227, 229-237, and U.S. Pat.Nos. 5,814,618 and 5,789,156, Lac repressor system promoters,copper-inducible system promoters, salicylate-inducible system promoters(e.g., the PR1a system), glucocorticoid-inducible promoters (Aoyama etal. (1997) Plant J. 11:605-612), and ecdysone-inducible systempromoters.

Other non-limiting examples of inducible promoters include ABA- andturgor-inducible promoters, the auxin-binding protein gene promoter(Schwob et al. (1993) Plant J. 4:423-432), the UDP glucose flavonoidglycosyl-transferase promoter (Ralston et al. (1988) Genetics119:185-197), the MPI proteinase inhibitor promoter (Cordero et al.(1994) Plant J. 6:141-150), and the glyceraldehyde-3-phosphatedehydrogenase promoter (Kohler et al. (1995) Plant Mol. Biol.29:1293-1298; Martinez et al. (1989) J. Mol. Biol. 208:551-565; andQuigley et al. (1989) J. Mol. Evol. 29:412-421). Also included are thebenzene sulphonamide-inducible (U.S. Pat. No. 5,364,780) andalcohol-inducible (Int'l Patent Application Publication Nos. WO 97/06269and WO 97/06268) systems and glutathione S-transferase promoters.Likewise, one may use any of the inducible promoters described in Gatz(1996) Current Opinion Biotechnol. 7:168-172 and Gatz (1997) Annu. Rev.Plant Physiol. Plant Mol. Biol. 48:89-108. Other chemically induciblepromoters useful for directing the expression of the nucleotidesequences of this invention in plants are disclosed in U.S. Pat. No.5,614,395 herein incorporated by reference in its entirety. Chemicalinduction of gene expression is also detailed in the publishedapplication EP 0 332 104 (to Ciba-Geigy) and U.S. Pat. No. 5,614,395. Insome embodiments, a promoter for chemical induction may be the tobaccoPR-1a promoter.

In some embodiments, promoters useful with algae include, but are notlimited to, the promoter of the RubisCo small subunit gene 1 (PrbcS1),the promoter of the actin gene (Pactin), the promoter of the nitratereductase gene (Pnr) and the promoter of duplicated carbonic anhydrasegene 1 (Pdca1) (See, Walker et al. Plant Cell Rep. 23:727-735 (2005); Liet al. Gene 403:132-142 (2007); Li et al. Mol Biol. Rep. 37:1143-1154(2010)), the promoter of the σ⁷⁰-type plastid rRNA gene (Prrn), thepromoter of the psbA gene (encoding the photosystem-II reaction centerprotein D1) (PpsbA), the promoter of the psbD gene (encoding thephotosystem-II reaction center protein D2) (PpsbD), the promoter of thepsaA gene (encoding an apoprotein of photosystem I) (PpsaA), thepromoter of the ATPase alpha subunit gene (PatpA), and promoter of theRuBisCo large subunit gene (PrbcL), and any combination thereof (See,e.g., De Cosa et al. Nat. Biotechnol. 19:71-74 (2001); Daniell et al.BMC Biotechnol. 9:33 (2009); Muto et al. BMC Biotechnol. 9:26 (2009);Surzycki et al. Biologicals 37:133-138 (2009)).

In some embodiments, promoters useful with bacteria and yeast include,but are not limited to, a constitutive promoter (e.g., lpp (lipoproteingene)) and/or an oxidative stress inducible promoter (e.g., a superoxidedismutase or a catalase promoter).

Thus, in some embodiments, a promoter useful with yeast may include, butis not limited to, a promoter from phosphoglycerate kinase (PGK),glyceraldehyde-3-phosphate dehydrogenase (GAP), triose phosphateisomerase (TPJ), galactose-regulon (GAL1, GAL10), alcohol dehydrogenase(ADH1, ADH2), phosphatase (PHO5), copper-activated metallothionine(CUP1), MFα1, PGK/α2 operator, TPI/α2 operator, GAP/GAL, PGK/GAL,GAP/ADH2, GAP/PHO5, iso-1-cytochrome c/glucocorticoid response element(CYC/GRE), phosphoglycerate kinase/angrogen response element (PGK/ARE),transcription elongation factor EF-1α (TEF1), triose phosphatedehydrogenase (TDH3), phosphoglycerate kinase 1 (PGK1), pyruvate kinase1 (PYK1), and/or hexose transporter (HXT7) (See, Romanos et al. Yeast8:423-488 (1992); and Partow et al. Yeast 27:955-964 (2010)).

In some embodiments, a promoter useful with bacteria may include, but isnot limited to, L-arabinose inducible (araBAD, P_(BAD)) promoter, anylac promoter, L-rhamnose inducible (rhaP_(BAD)) promoter, T7 RNApolymerase promoter, trc promoter, tac promoter, lambda phage promoter(p_(L), p_(L)-9G-50), anydrotetracycline-inducible (tetA) promoter, trp,lpp, phoA, recA, proU, cst-1, cadA, nar, lpp-lac, cspA, T7-lac operator,T3-lac operator, T4 gene 32, T5-lac operator, nprM-lac operator, Vhb,Protein A, corynebacterial-E. coli like promoters, thr, homrn,diphtheria toxin promoter, sig A, sig B, nusG, SoxS, katb, α-amylase(Pamy), Ptms, P43 (comprised of two overlapping RNA polymerase σ factorrecognition sites, σA, σB), Ptms, P43, rplK-rplA, ferredoxin promoter,and/or xylose promoter. (See, K. Terpe Appl. Microbiol, Biotechnol.72:211-222 (2006); Hannig et al. Trends in Biotechnology 16:54-60(1998); and Srivastava et al., Protein Expr Purif 40:221-229 (2005)).

As used herein, “expression cassette” means a recombinant nucleic acidmolecule comprising a nucleotide sequence encoding a modified Vip3polypeptide of the invention, wherein said nucleotide sequence isoperably associated with at least a control sequence (e.g., a promoter).Thus, some aspects of the invention provide expression cassettesdesigned to express the nucleotides sequences encoding the modified Vip3polypeptides of the invention.

An expression cassette comprising a nucleotide sequence of interest maybe chimeric, meaning that at least one of its components is heterologouswith respect to at least one of its other components. An expressioncassette may also be one that is naturally occurring but has beenobtained in a recombinant form useful for heterologous expression.

In addition to the promoters operatively linked to the nucleotidesequences of the invention, an expression cassette of this inventionalso may include other regulatory sequences. Thus, an expressioncassette also may optionally include a transcriptional and/ortranslational termination region (i.e., termination region) that isfunctional in the selected host cell. A variety of transcriptionalterminators are available for use in expression cassettes and areresponsible for the termination of transcription beyond the heterologousnucleotide sequence of interest and correct mRNA polyadenylation. Thetermination region may be native to the transcriptional initiationregion, may be native to the operably linked nucleotide sequence ofinterest, may be native to the host cell, or may be derived from anothersource (i.e., foreign or heterologous to the promoter, to the nucleotidesequence of interest, to the host, or any combination thereof).Appropriate transcriptional terminators are those that are known tofunction in the host cell of interest. For plants, such terminators mayinclude but are not limited to the CAMV 35S terminator, the tmlterminator, the nopaline synthase terminator, and the pea rbcs E9terminator.

Numerous nucleotide sequences have been found to enhance gene expressionfrom within the transcriptional unit and these sequences may be used inconjunction with the expression cassettes of this invention to increasethe expression of a polynucleotide of interest in a host cell.

Various intron sequences have been shown to enhance expression,particularly in monocotyledonous cells. For example, intron sequencesare routinely incorporated into plant transformation vectors, typicallywithin the non-translated leader.

A number of non-translated leader sequences derived from viruses arealso known to enhance expression, and these are encompassed herein.Specifically, leader sequences from Tobacco Mosaic Virus (TMV, the“Ω-sequence”), Maize Chlorotic Mottle Virus (MCMV), and Alfalfa MosaicVirus (AMV) have been shown to be effective in enhancing expression(see, for example, Gallie et al. (1987) Nucleic Acids Res. 15:8693-8711;Skuzeski et al. (1990) Plant Molec. Biol. 15:65-79). Other leadersequences known in the art include, but are not limited to, picomavirusleaders, for example, EMCV leader (Encephalomyocarditis 5′ noncodingregion) (Elroy-Stein et al. (1989) Proc. Natl. Acad. Sci. USA86:6126-6130); potyvirus leaders, for example, TEV leader (Tobacco EtchVirus) (Allison et al. (1986) Virology 154:9-20; and Gallie et al.(1995) Gene 165:233-238); MDMV leader (Maize Dwarf Mosaic Virus; Allisonet al. (1986) Virology 154:9-20); human immunoglobulin heavy-chainbinding protein (BiP) leader (Macejak and Samow (1991) Nature353:90-94); untranslated leader from the coat protein mRNA of alfalfamosaic virus (AMV RNA 4) (Jobling et al. (1987) Nature 325:622-625);tobacco mosaic virus leader (TMV; Gallie et al. (1987) Nucleic AcidsRes. 15:3257-3273; Gallie et al. (1988) Nucleic Acids Res. 16:883-893;Gallie et al. (1992) Nucleic Acids Res. 20:4631-4638); and MaizeChlorotic Mottle Virus leader (MCMV; Lommel et al. (1991) Virology81:382-385). See also, Della-Cioppa et al. (1987) Plant Physiology84:965-968.

An expression cassette also may include a nucleotide sequence for aselectable marker, which may be used to select a transformed host cell.As used herein, “selectable marker” means a nucleotide sequence thatwhen expressed imparts a distinct phenotype to the host cell expressingthe marker and thus allows such transformed cells to be distinguishedfrom those that do not have the marker. Such a nucleotide sequence mayencode either a selectable or screenable marker, depending on whetherthe marker confers a trait that may be selected for by chemical means,such as by using a selective agent (e.g., an antibiotic and the like),or on whether the marker is simply a trait that one can identify throughobservation or testing, such as by screening (e.g., fluorescence). Ofcourse, many examples of suitable selectable markers are known in theart and may be used in the expression cassettes described herein.

In addition to expression cassettes, the nucleic acid molecules andnucleotide sequences described herein may be used in connection withvectors. The term “vector” refers to a composition for transferring,delivering or introducing a nucleic acid (or nucleic acids) into a cell.A vector comprises a nucleic acid molecule comprising the nucleotidesequence(s) to be transferred, delivered or introduced. Vectors for usein transformation of host organisms are well known in the art.Non-limiting examples of general classes of vectors include but are notlimited to a viral vector, a plasmid vector, a phage vector, a phagemidvector, a cosmid vector, a fosmid vector, an artificial chromosome, oran Agrobacterium binary vector in double or single stranded linear orcircular form which may or may not be self transmissible or mobilizable.A vector as defined herein may transform prokaryotic or eukaryotic hosteither by integration into the cellular genome or existextrachromosomally (e.g. autonomous replicating plasmid with an originof replication). Additionally included are shuttle vectors by which ismeant a DNA vehicle capable, naturally or by design, of replication intwo different host organisms, which may be selected from actinomycetesand related species, bacteria and eukaryotic (e.g., higher plants,mammals, fungi, including yeast) organisms. In some embodiments, thenucleic acid in the vector is under the control of, and operably linkedto, an appropriate promoter or other regulatory elements fortranscription in a host cell. The vector may be a bi-functionalexpression vector which functions in multiple hosts. In the case ofgenomic DNA, this may contain its own promoter or other regulatoryelements and in the case of cDNA this may be under the control of anappropriate promoter or other regulatory elements for expression in thehost cell. Accordingly, the nucleic acid molecules of this inventionand/or expression cassettes may be comprised in vectors as describedherein and as known in the art.

In some embodiments, it may be desirable to target the modified Vip3polypeptides of the invention to particular parts of a cell such as thechloroplast, the cell wall, the mitochondria, and the like. A nucleotidesequence encoding a signal peptide may be operably linked at the 5′- or3′-terminus of a heterologous nucleotide sequence or nucleic acidmolecule.

Various mechanisms for targeting gene products are known to exist inplants and the sequences controlling the functioning of these mechanismshave been characterized in some detail. For example, the targeting ofgene products to the chloroplast is controlled by a signal sequencefound at the amino terminal end of various proteins, which is cleavedduring chloroplast import to yield the mature protein (see, e.g., Comaiet al. (1988) J. Biol. Chem. 263:15104-15109). These signal sequencesmay be fused to heterologous gene products to effect the import ofheterologous products into the chloroplast (van den Broeck et al. (1985)Nature 313:358-363). DNA encoding for appropriate signal sequences maybe isolated from the 5′ end of the cDNAs encoding the RUBISCO protein,the CAB protein, the EPSP synthase enzyme, the GS2 protein and manyother proteins that are known to be chloroplast localized. See also, thesection entitled “Expression with Chloroplast Targeting” in Example 37of U.S. Pat. No. 5,639,949.

The above-described targeting sequences may be utilized not only inconjunction with their endogenous promoters, but also in conjunctionwith heterologous promoters. Use of promoters that are heterologous tothe targeting sequence not only provides the ability to target thesequence but also can provide an expression pattern that is differentfrom that of the promoter from which the targeting signal is originallyderived.

Thus, signal peptides (and the targeting nucleotide sequences encodingthem) are well known in the art and can be found in public databasessuch as the “Signal Peptide Website: An Information Platform for SignalSequences and Signal Peptides.” the “Signal Peptide Database” (Choo etal., BMC Bioinformatics 6:249 (2005); ChloroP predicts the presence ofchloroplast transit peptides (cTP) in protein sequences and the locationof potential cTP cleavage sites); LipoP predicts lipoproteins and signalpeptides in Gram negative bacteria); MITOPROT predicts mitochondrialtargeting sequences); PlasMit predicts mitochondrial transit peptides inPlasmodium falciparum); Predotar predicts mitochondrial and plastidtargeting sequences); PTS1 predicts peroxisomal targeting signal 1containing proteins); SignalP predicts the presence and location ofsignal peptide cleavage sites in amino acid sequences from differentorganisms: Gram-positive prokaryotes, Gram-negative prokaryotes, andeukaryotes).

Thus, for example, to localize to a plastid a transit peptide fromplastidic Ferredoxin: NADP+ oxidoreductase (FNR) of spinach, which isdisclosed in Jansen et al. (1988) Current Genetics 13:517-522, may beemployed. In particular, the sequence ranging from the nucleotides −171to 165 of the cDNA sequence disclosed therein may be used, whichcomprises the 5′ non-translated region as well as the sequence encodingthe transit peptide. Another example of a transit peptide is that of thewaxy protein of maize including the first 34 amino acid residues of themature waxy protein (Klosgen et al. (1989) Mol. Gen. Genet.217:155-161). It is also possible to use this transit peptide withoutthe first 34 amino acids of the mature protein. Furthermore, the signalpeptides of the ribulose bisphosphate carboxylase small subunit (Wolteret al. (1988) Proc. Natl. Acad. Sci. USA 85:846-850; Nawrath et al.(1994) Proc. Natl. Acad. Sci. USA 91:12760-12764), of NADP malatedehydrogenase (Galiardo et al. (1995) Planta 197:324-332), ofglutathione reductase (Creissen et al. (1995) Plant J. 8:167-175) and/orof the R1 protein (Lorberth et al. (1998) Nature Biotechnology16:473-477) may be used.

“Introducing,” “introduce,” “introduced” (and grammatical variationsthereof) in the context of a polynucleotide of interest means presentingthe nucleotide sequence of interest to the host organism or cell of saidorganism (e.g., host cell) in such a manner that the nucleotide sequencegains access to the interior of a cell. Where more than one nucleotidesequence is to be introduced these nucleotide sequences may be assembledas part of a single polynucleotide or nucleic acid construct, or asseparate polynucleotide or nucleic acid constructs, and may be locatedon the same or different expression constructs or transformationvectors. Accordingly, these polynucleotides may be introduced into cellsin a single transformation event, in separatetransformation/transfection events, or, for example, they may beincorporated into an organism by conventional breeding protocols (e.g.,crosses). Thus, in some aspects of the present invention one or morenucleic acid constructs of this invention (e.g., a nucleic acid moleculecomprising a nucleotide sequence encoding a modified Vip3 polypeptide ofthe invention) may be introduced into a host organism or a cell of saidhost organism.

The term “transformation” or “transfection” as used herein refers to theintroduction of a heterologous nucleic acid into a cell. Transformationof a cell may be stable or transient. Thus, in some embodiments, a hostcell or host organism is stably transformed with a nucleic acid moleculeof the invention. In some embodiments, a host cell or host organism istransiently transformed with a recombinant nucleic acid molecule of theinvention.

“Transient transformation” in the context of a polynucleotide means thata polynucleotide is introduced into the cell and does not integrate intothe genome of the cell.

“Stable transformation” or “stably transformed” as used herein meansthat a nucleic acid molecule is introduced (e.g., “stably introducing”or “stably introduced”) into a cell and integrates into the genome ofthe cell. As such, the integrated nucleic acid molecule is capable ofbeing inherited by the progeny thereof, more particularly, by theprogeny of multiple successive generations. “Genome” as used herein alsoincludes the nuclear and the plastid genome, and therefore includesintegration of the nucleic acid into, for example, the chloroplast ormitochondrial genome. Stable transformation as used herein may alsorefer to a transgene that is maintained extrachromasomally, for example,as a minichromosome or a plasmid.

Transient transformation may be detected by, for example, anenzyme-linked immunosorbent assay (ELISA) or Western blot, which candetect the presence of a peptide or polypeptide encoded by one or moretransgene introduced into an organism. Stable transformation of a cellmay be detected by, for example, a Southern blot hybridization assay ofgenomic DNA of the cell with nucleic acid sequences which specificallyhybridize with a nucleotide sequence of a transgene introduced into anorganism (e.g., a plant, a mammal, an insect, an archaea, a bacterium,and the like). Stable transformation of a cell may be detected by, forexample, a Northern blot hybridization assay of RNA of the cell withnucleic acid sequences which specifically hybridize with a nucleotidesequence of a transgene introduced into a plant or other organism.Stable transformation of a cell may also be detected by, e.g., apolymerase chain reaction (PCR) or other amplification reactions as arewell known in the art, employing specific primer sequences thathybridize with target sequence(s) of a transgene, resulting inamplification of the transgene sequence, which may be detected accordingto standard methods Transformation may also be detected by directsequencing and/or hybridization protocols well known in the art.

Accordingly, in some embodiments, the nucleic acid molecule, nucleotidesequences, constructs, expression cassettes may be expressed transientlyand/or they may be stably incorporated into the genome of the hostorganism.

A recombinant nucleic acid molecule/polynucleotide of the invention maybe introduced into a cell by any method known to those of skill in theart. In some embodiments of the invention, transformation of a cellcomprises nuclear transformation. In some embodiments, transformation ofa cell comprises plastid transformation (e.g., chloroplasttransformation). In some embodiments, the recombinant nucleic acidmolecule/polynucleotide of the invention may be introduced into a cellvia conventional breeding techniques (e.g., crossing).

Procedures for transforming both eukaryotic and prokaryotic organismsare well known and routine in the art and are described throughout theliterature (See, for example, Jiang et al. 2013. Nat. Biotechnol.31:233-239; Ran et al. Nature Protocols 8:2281-2308 (2013))

A nucleotide sequence therefore may be introduced into a host organismor its cell in any number of ways that are well known in the art. Themethods of the invention do not depend on a particular method forintroducing one or more nucleotide sequences into the organism, onlythat they gain access to the interior of at least one cell of theorganism. Where more than one nucleotide sequence is to be introduced,they may be assembled as part of a single nucleic acid construct, or asseparate nucleic acid constructs, and may be located on the same ordifferent nucleic acid constructs. Accordingly, the nucleotide sequencesmay be introduced into the cell of interest in a single transformationevent, or in separate transformation events, or, alternatively, whererelevant, a nucleotide sequence may be incorporated into a plant, aspart of a breeding protocol.

In some embodiments, a nucleic acid construct, a nucleic acid molecule,and/or a nucleotide sequence of this invention may be introduced into acell of a host organism. Any cell/host organism for which this inventionis useful with may be used. Exemplary host organisms include a plant, abacterium, an archaeon, a virus, an animal (e.g., an insect), and/or afungus (e.g., a yeast).

As used herein, “plant” means any plant and thus includes, for example,angiosperms including both monocots and dicots, gymnosperms, bryophytes,ferns and/or fern allies. In some embodiments of this invention, theplant is a seed plant. Further, a “plant” of this invention is any plantat any stage of development.

As used herein, the term “plant part” or “plant material” includes butis not limited to embryos, pollen, ovules, seeds, leaves, stems, roots,flowers or flower parts, branches, fruit, kernels, ears, cobs, husks,stalks, roots, root tips, anthers, pollen, egg cells, zygotes, cuttings,plant cells including plant cells that are intact in plants and/or partsof plants, plant protoplasts, plant tissues, plant cell tissue cultures,plant calli, plant clumps, or any other part or product of a plant.Further, as used herein, “plant cell” refers to a structural andphysiological unit of the plant comprising a protoplast and a cell wall.Thus, in some embodiments, a plant cell of the invention may be in theform of an isolated single cell or may be a cultured cell or may be apart of a higher-organized unit such as, for example, a plant tissue ora plant organ.

A “protoplast” is an isolated plant cell without a cell wall or withonly parts of the cell wall.

“Plant cell culture” means cultures of plant units such as, for example,protoplasts, cell culture cells, cells in plant tissues, pollen, pollentubes, ovules, embryo sacs, zygotes and embryos at various stages ofdevelopment.

As used herein, a “plant organ” is a distinct and visibly structured anddifferentiated part of a plant such as a root, stem, leaf, flower bud,or embryo.

“Plant tissue” as used herein means a group of plant cells organizedinto a structural and functional unit. Any tissue of a plant in plantaor in culture is included. This term includes, but is not limited to,whole plants, plant organs, plant seeds, tissue culture and any groupsof plant cells organized into structural and/or functional units. Theuse of this term in conjunction with, or in the absence of, any specifictype of plant tissue as listed above or otherwise embraced by thisdefinition is not intended to be exclusive of any other type of planttissue.

The present invention is directed to modified vegetative insecticidalproteins (Vip), compositions comprising the same and methods of usethereof. In some embodiments, the Vip polypeptide is Vip3 polypeptide.

Vip3 polypeptides and nucleic acids encoding the same are known anddescribed in, for example, J. Estruch et al., Proc. Natl. Acad. Sci. USA93, 5389-5394 (May 1996); J. Liu et al., Letters in Applied Microbiology45, 432-438 (2007); G. Warren, U.S. Pat. No. 5,990,383; Z. Shen et al.,U.S. Pat. No. 7,378,493; P. Miles et al., U.S. Pat. Nos. 7,244,820;5,877,012; 6,107,279, 6,137,033, and 6,291,156, the disclosures of allof which are incorporated by reference herein in their entirety. Thenaming of Vip proteins by the research groups discovering the respectiveproteins has varied. However, a standardized nomenclature has recentlybeen developed and can be found at the Bacillus thuringiensis ToxinNomenclature database. The present disclosure specifically exemplifiesmodifications to a Vip3A, Vip3B, Vip3C and Vip3D polypeptides, but thepresent invention may be practiced with any Vip polypeptide (e.g., anyVip3 polypeptide) now known or later discovered.

Accordingly, a “Vip3 polypeptide” in the context of the invention meansany vegetative insecticidal protein (VIP) now or later identified thatis a member of the Vip3 class including, for example, withoutlimitation, Vip3A, Vip3B, Vip3C, Vip3D, and their homologues. In someembodiments, a Vip3 polypeptide useful with this invention can be amutant Vip3 that is naturally occurring or non-naturally occurring. Somestructural features that identify a protein as being in the Vip3 classof proteins includes, 1) a size of about 80-88 kDa that isproteolytically processed by insects or trypsin to about a 62-66 kDatoxic core (Lee et al. 2003. Appl. Environ. Microbiol. 69:4648-4657);and 2) a highly conserved N-terminal secretion signal which is notnaturally processed during secretion in B. thuringiensis. Non-limitingexamples of members of the Vip3 class including those previouslymentioned and their respective GenBank accession numbers, U.S. patent orpatent publication number are Vip3Aa1 (AAC37036), Vip3Aa2 (AAC37037),Vip3Aa3 (U.S. Pat. No. 6,137,033), Vip3Aa4 (AAR81079), Vip3Aa5(AAR81080), Vip3Aa6 (AAR81081), Vip3Aa7 (AAK95326), Vip3Aa8 (AAK97481),Vip3Aa9 (CAA76665), Vip3Aa10 (AAN60738), Vip3Aa11 (AAR36859), Vip3Aa12(AAM22456), Vip3Aa13 (AAL69542), Vip3Aa14 (AAQ12340), Vip3Aa15(AAP51131), Vip3Aa16 (AAW65132), Vip3Aa17 (U.S. Pat. No. 6,603,063),Vip3Aa18 (AAX49395), Vip3Aa19 (DQ241674), Vip3Aa19 (DQ539887), Vip3Aa20(DQ539888), Vip3Aa21 (ABD84410), Vip3Aa22 (AAY41427), Vip3Aa23(AAY41428), Vip3Aa24 (BI 880913), Vip3Aa25 (EF608501), Vip3Aa26(EU294496), Vip3Aa27 (EU332167), Vip3Aa28 (FJ494817), Vip3Aa29(FJ626674), Vip3Aa30 (FJ626675), Vip3Aa31 (FJ626676), Vip3Aa32(FJ626677), Vip3Aa33 (GU073128), Vip3Aa34 (GU073129), Vip3Aa35(GU733921), Vip3Aa36 (GU951510), Vip3Aa37 (HM132041), Vip3Aa38(HM117632), Vip3Aa39 (HM117631), Vip3Aa40 (HM132042), Vip3Aa41(HM132043), Vip3Aa42 (HQ587048), Vip3Aa43 (HQ594534), Vip3Aa44(HQ650163), Vip3Ab1 (AAR40284), Vip3Ab2 (AAY88247), Vip3Ac1 (U.S. PatentApplication Publication 20040128716), Vip3Ad1 (U.S. Patent ApplicationPublication 20040128716), Vip3Ad2 (CAI43276), Vip3Ae1 (CAI43277),Vip3Af1 (U.S. Pat. No. 7,378,493), Vip3Af2 (ADN08753), Vip3Af3(HM117634), Vip3Ag1 (ADN08758), Vip3Ag2 (FJ556803), Vip3Ag3 (HM117633),Vip3Ag4 (HQ414237), Vip3Ag5 (HQ542193), Vip3Ah1 (DQ832323), Vip3Ba1(AAV70653), Vip3Ba2 (HM117635), Vip3Bb1 (U.S. Pat. No. 7,378,493),Vip3Bb2 (ABO030520), Vip3C (Palma et al. Appl. Environ Microbiol78(19):7163-7165 (2012)) and/or Vip3Bb3 (ADI48120).

The present inventors have surprisingly discovered that modifying a Vip3polypeptide such that it comprises a heterologous carbohydrate bindingmodule (CBM) results in a modified Vip3 polypeptide having alteredcharacteristics including altered toxicity toward plant pests ascompared to the same Vip3 polypeptide that is not modified to comprisesaid heterologous CBM (i.e., a reference Vip3).

Accordingly, in one aspect of the invention a modified Vip3 polypeptidecomprising, consisting essentially of, or consisting of a heterologouscarbohydrate binding module (CBM) is provided. In some embodiments, amodified Vip 3 polypeptide can comprise, consist essentially of, orconsist of two or more CBMs, which can be the same or different,optionally in tandem. In some embodiments, the heterologous CBM may besubstituted for all or a portion of Domain III of a Vip3 polypeptide. Insome embodiments, the modified Vip3 polypeptide comprises all or aportion of Domain I and/or Domain II of a Vip3 polypeptide. In someembodiments, the modified Vip3 polypeptide may comprise, consistessentially of, or consist of all or a portion of Domain IV of a Vip3polypeptide and/or may lack all or a portion of Domain IV of a Vip3polypeptide. In some embodiments, the modified Vip3 polypeptidecomprises, consists essentially of, or consists of, in the aminoterminal to carboxy terminal direction, all or a portion of Domain I ofthe Vip3 polypeptide, all or a portion of Domain II of the Vip3polypeptide, the heterologous CBM, and optionally all or a portion ofDomain IV of the Vip3 polypeptide.

In some embodiments, the modified Vip3 polypeptide of the inventioncomprises, consists essentially of, or consists of all or a portion ofany one of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, or SEQ IDNO:6 or an amino acid sequence having at least 70% identity to saidportion of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, or SEQ IDNO:6.

In some embodiments, Domain III of a Vip3 polypeptide comprises,consists essentially of, or consists of amino acids 518 to 684 of saidVip3 polypeptide. In particular embodiments, Domain III comprises,consists essentially of, or consists of amino acids 542 to 667 of SEQ IDNOs:1-3 or a corresponding amino acid sequence from a different Vip3polypeptide, amino acids 550 to 675 of SEQ ID NO:4 or a correspondingamino acid sequence from a different Vip3 polypeptide, or amino acids552 to 667 of SEQ ID NO:6 or a corresponding amino acid sequence from adifferent Vip3 polypeptide.

“Corresponding to” in the context of the present invention means thatwhen the amino acid sequences of certain proteins are aligned with eachother, the amino acids that “correspond to” certain enumerated positionsin the present invention are those that align with these positions in areference sequence, but that are not necessarily in these exactnumerical positions relative to a particular amino acid sequence of theinvention.

In some embodiments, Domain III of a Vip3 polypeptide comprises,consists essentially of, or consists of:

(a) amino acids 542 to 667 of SEQ ID NO:1;

(b) amino acids 542 to 667 of SEQ ID NO:2;

(c) amino acids 542 to 667 of SEQ ID NO:3;

(d) amino acids 550 to 675 of SEQ ID NO:4;

(e) amino acids 552 to 677 of SEQ ID NO:6;

(f) a corresponding amino acid sequence of another Vip3 polypeptide asdescribed herein; or

(g) an amino acid sequence having at least about 70% identity to any oneof (a) to (f), above.

The heterologous CBM may be incorporated into the Vip3 polypeptide, forexample, by insertion or by substitution of a portion of the Vip3polypeptide. In embodiments in which the CBM in substituted for aportion of a Vip3 polypeptide, the portion of the Vip3 polypeptide thatis exchanged for a heterologous CBM may include all of Domain III, aportion of Domain III (e.g., fewer amino acid residues than the entiretyof Domain III) or may be a portion of the Vip3 polypeptide that isgreater than the entirety of Domain III (e.g., may extend in theN-terminal direction outside of Domain III and toward or into Domain IIand/or in the C-terminal direction outside of Domain III and toward orinto Domain IV). Thus, for example, when Domain III of a Vip3polypeptide is swapped or exchanged for a heterologous CBM, one or moreamino acid residues (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, or more residues) of the Vip3 polypeptide,at the amino terminal side and/or the carboxy terminal side of DomainIII, also may be included in the exchange. Thus, for example, whenDomain III comprises amino acids 542 to 667 of SEQ ID NO:1, thesubstitution may include one or more of amino acid residues 500 to 541(e.g., amino acid 500, 501, 502, 503, 504, 505, 506, 507, 508, 509, 510,511, 512, 513, 514, 515, 516, 517, 518, 519, 520, 522, 523, 524, 525,526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 537, 538, 539,540, 541 or any range therein) at the amino terminal side and/or one ormore of amino acids 668 to 700 (e.g., amino acid 668, 669, 670, 671,672, 673, 674, 675, 676, 678, 679, 680, 681, 682, 683, 684, 685, 686,687, 688, 689, 690, 691, 692, 693, 694, 695, 696, 697, 698, 699, 700 orany range therein) at the carboxy terminal side of said Domain III, inany combination. In some embodiments, the substitution can comprise,consist essentially of, or consist of portion of Domain III that isabout one to about four (e.g., 1, 2, 3, or 4) amino acid residuesshorter than the full Domain III at the C-terminal end and/or theN-terminal end. In some embodiments, the substitution can comprise,consist essentially of, or consist of Domain III and an additional oneto about ten (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acidresidues more than the full Domain III at the C-terminal end and/or theN-terminal end.

In some embodiments, at least a portion of a Vip3 Domain III asdescribed herein may be exchanged for a CBM. Thus, for example, whenDomain III is amino acids 550 to 675 of SEQ ID NO:4, the substitutionmay include fewer than all of the amino acids residues 550 to 675 byretaining in the Vip3 polypeptide one or more of the amino acid residues(e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, or more residues, and the like) at the amino terminal sideand/or the carboxy terminal side of Domain III of SEQ ID NO:4 (e.g.,amino acid residues 550 to 675). As an example, for SEQ ID NO:4, theportion of Domain III that is exchanged may be the full length aminoacid sequence of residues 550 to 675, or it may be, for example,residues 551 to 675, 552 to 675, 553 to 675, 554 to 675, 551 to 674, 551to 673, 551 to 672, 550 to 673, 555 to 670, 560 to 675, 560 to 670, andso on.

Thus, the region of the Vip3 polypeptide that is exchanged for theheterologous CBM may be any combination of all of Domain III, or moreand/or fewer amino acid residues at the carboxy terminus and/or theamino terminus of Domain III of the Vip3 polypeptide.

In some embodiments, a modified Vip3 polypeptide of the invention has atleast about 70% (e.g., 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%,80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, 100%) amino acid sequence similarity oridentity with an amino acid sequence of SEQ ID NO:1, SEQ ID NO:2, SEQ IDNO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8,SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ IDNO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ IDNO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ IDNO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ IDNO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ IDNO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ IDNO:53, SEQ ID NO:54, SEQ ID NO:55, or SEQ ID NO:56 (i.e., SEQ IDNOs:1-23 or SEQ ID NOs:33-56). In some embodiments, a modified Vip3polypeptide of the invention has at least about 75% amino acid sequencesimilarity or identity with an amino acid sequence selected from any oneof SEQ ID NOs:1-23 or SEQ ID NOs:33-56. In some embodiments, a modifiedVip3 polypeptide of the invention has at least about 80% amino acidsequence similarity or identity with an amino acid sequence selectedfrom any one of SEQ ID NOs:1-23 or SEQ ID NOs:33-56. In someembodiments, a modified Vip3 polypeptide of the invention has at leastabout 85% amino acid sequence similarity or identity with an amino acidsequence selected from any one of SEQ ID NOs:1-23 or SEQ ID NOs:33-56.In some embodiments, a modified Vip3 polypeptide of the invention has atleast about 90% amino acid sequence similarity or identity with an aminoacid sequence selected from any one of SEQ ID NOs:1-23 or SEQ IDNOs:33-56. In some embodiments, a modified Vip3 polypeptide of theinvention has at least about 95% amino acid sequence similarity oridentity with an amino acid sequence selected from any one of SEQ IDNOs:1-23 or SEQ ID NOs:33-56. In some embodiments, a modified Vip3polypeptide of the invention has at least about 90 to about 100% aminoacid sequence similarity or identity with an amino acid sequenceselected from any one of SEQ II NOs:1-23 or SEQ ID NOs:33-56. In someembodiments, a modified Vip3 polypeptide of the invention has 100% aminoacid sequence similarity or identity with an amino acid sequenceselected from any one of SEQ ID NOs:1-23 or SEQ ID NOs:33-56.

In representative embodiments, a modified Vip3 polypeptide of theinvention may be an “improved Vip3 polypeptide” when compared to itswild-type or reference parent Vip3 polypeptide, in that it displays oneor more of the following characteristics: 1) an increased potencyagainst a target insect (higher specific activity) and/or an increasedkill rate (faster kill at comparable level of protein); 2) increased ordecreased target pest spectrum; 3) decreased susceptibility todevelopment of resistance by target pests; 4) increased expressionlevels in a transgenic host or host cell; 5) increased resistance toinsect protease degradation (increased stability in the target insectgut); 6) increased stability in the environment; and 7) reduced toxicityto beneficial insects, non-target pests, and plants.

Therefore, in the context of the invention, “improves pesticidal (e.g.,insecticidal, nematicidal) activity” or “improved pesticidal (e.g.,insecticidal) activity,” or any grammatical variation thereof, meansthat a modification of the Vip3 polypeptide results in an engineeredpolypeptide of the invention having one or more of the followingcharacteristics: 1) an increased potency against a target pest (e.g.,insect) (i.e., higher specific activity) and/or an increased kill rate(faster kill at comparable level of protein), 2) increased or decreasedtarget pest spectrum, 3) decreased susceptibility to development ofresistance by target pests, 4) increased expression levels in atransgenic host or host cell, 5) increased resistance to insect proteasedegradation (increased stability in the target insect gut), 6) increasedstability in the environment and 7) reduced toxicity to beneficialinsects, non-target pests, and plants.

Carbohydrate binding modules (CBMs) are discretely folded domains thatare found within a protein that is a carbohydrate active enzyme. Astheir name indicates, CBMs are characterized by their carbohydratebinding activity (see, e.g., Boraston et al. Biochem J. 382:769-781)(2004)). CBMs are non-catalytic domains connected to catalytic modulesin the larger polypeptide via linker sequences that are sometimes highlyflexible (Gilbert et al. Curr. Op. Structural Biol. 23; 669-677 (2013)).It is generally believed that CBMs function to bring the enzyme of whichthey are a part in closer proximity with the target substrate, therebyleading to an increase in the rate of catalysis.

Currently, carbohydrate-binding modules are classified into 67 familiesbased on amino acid sequence similarities (see, Carbohydrate ActiveenZyme database Cantarel et al. (2009). “The Carbohydrate-Active EnZymesdatabase (CAZy): An expert resource for Glycogenomics”. Nucleic AcidsResearch 37 (Database issue): D233-D238.

A heterologous CBM useful with this invention may be a Type B CBM fromany protein. Type B CBMs are glycan chain binding CBMs having grooves orclefts. (Boraston et al. Biochem J (382:769-781 (2004)). In someembodiments, a Type B CBM useful with this invention comprises aβ-sandwich. In some embodiments, a heterologous CBM comprises, consistsessentially of, or consists of a CBM amino acid sequence from aβ-1,4-mannanase, optionally a Type B CBM amino acid sequence from aβ-1,4-mannanase.

In some embodiments, a CBM useful with the invention is from aβ-1,4-mannanase. The EC number provided by the International Union ofBiochemistry and Molecular Biology (IUBMB) for this enzyme family is EC3.2.1.78. Thus, in some embodiments, a CBM useful with this invention isan enzyme from the enzyme class EC 3.2.1.78. The accepted name for thisclass of enzymes is mannan endo-1,4-beta-mannosidase but these enzymesare also known by the names of β-1,4-mannanase, endo-1,4-mannanase,beta-mannanase; endo-1,4-β-mannanase, endo-β-1,4-mannanase, β-mannanaseB, β-1, 4-mannan 4-mannanohydrolase, endo-β-mannanase, β-D-mannanase,and/or 1,4-β-D-mannan mannanohydrolase.

In some embodiments, a modified Vip3 may comprise a functional portionof a heterologous CBM, such as a Type B CBM, or a Type B CBM from a1,4-β mannanase. A functional portion of a CBM may include any aminoacid residue that falls within approximately about 3 angstroms to about8 angstroms (E.g., about 3, 4, 5, 6, 7, 8 angstroms and the like) of thecarbohydrate binding site and/or influences the conformation of an aminoacid which interacts with the carbohydrate. Amino acid residues that mayinfluence the conformation of an amino acid that interacts with thecarbohydrate may include those that may make a hydrogen bond, a van derWaals interaction, a hydrophobic interaction, or a charge change withthe carbohydrate itself, water and/or an ion that directly interactswith the carbohydrate. Thus, in some embodiments, a heterologous CBMcomprises, consists essentially of, or consists of all or a functionalportion of a CBM amino acid sequence from a β-1,4-mannanase, optionallya Type B CBM amino acid sequence from a β-1,4-mannanase.

CBMs useful with this invention can be found, for example, in theCarbohydrate-Active enZYmes Database. Some non-limiting examples includeCBMs from: Mannan endo-1,4-beta-mannosidase from Caldicellulosiruptorobsidiansis OB47 (GenBank Acc. No. ADL41540.1); Mannanendo-1,4-beta-mannosidase from Paenibacillus mucilaginosus 3016 (GenBankAcc. No. AFC29293.1); beta-1,4-mannanase from Geobacillusstearothermophilus (GenBank Acc. No. AAC71692.1); or beta-1,4-mannanasefrom Vibrio sp. MA-138 (GenBank Acc. No. BAG69482.2).

Additional non-limiting examples of beta-1,4-mannanase polypeptides fromwhich the CBMs may be useful with this invention include a CBM from abeta-1,4-mannanase from: Caldicellulosiruptor saccharolyticus (GenBankAccession No. AAC44232.1); Vibrio sp. MA-138 (GenBank Accession No.BAG69482.2); Cellulosimicrobium sp. HY-13 (GenBank Accession No.AEE43708.1); Bacillus subtilis (GenBank Accession No. AEB98481.1);Haliotis discus discus (GenBank Accession No. BAI99559.1); Streptomycessp. s6-204 (GenBank Accession No. ABY90130.1); Vibrio sp. MA-138(GenBank Accession No. BAA25188.1); Klebsiella pneumoniae subsp.pneumoniae KPNIH27 (GenBank Accession No. AIA43525.1); Klebsiellaoxytoca (GenBank Accession No. AIE71926.1); Aeromonas caviae (GenBankAccession No. KEP91190.1); Klebsiella pneumoniae subsp. pneumoniaeKPR0928 (GenBank Accession No. AIE29885.1); Cronobacter pulveris (NCBIReference Sequence: WP_029591781.1); Gemmobacter nectariphilus (NCBIReference Sequence: WP_028029945.1); Thioalkalivibrio sp. ALJ24 (NCBIReference Sequence: WP_026287860.1); Paracoccus sp. N5 (NCBI ReferenceSequence: WP_026155388.1); Rhizobium sp. JGI 0001002-C21 (NCBI ReferenceSequence: WP_025570492.1); Cronobacter sakazakii (GenBank Accession No.KDP99185.1); Enterobacter asburiae (NCBI Reference Sequence:WP_024908493.1); Yersinia enterocolitica subsp. enterocolitica 8081(NCBI Reference Sequence: YP_001008241.1); Clostridium straminisolvensJCM 21531 (GenBank Accession No. GAE87707.1); or Vibrio furnissii NCTC11218 (GenBank: ADT88758.1). Other non-limiting examples ofbeta-1,4-mannanase polypeptides may be found at the Carbohydrate ActiveenZYmes (CAZY) database.

In some embodiments, a heterologous CBM comprises, consists essentiallyof, or consists of a CBM amino acid sequence that has at least about70%-100% (e.g., at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%,98%, 99%, 100%, or any variable or range therein) amino acid sequencesimilarity or identity with the amino acid sequence of a naturallyoccurring CBM, for example, a Type B CBM from a β 1,4-mannanase (e.g.,any one of SEQ ID NOs:24-32). In some embodiments, the heterologous CBMcomprises, consists essentially of, or consists of a CBM amino acidsequence that has at least about 75% amino acid sequence similarity oridentity with the amino acid sequence of any one of SEQ ID NOs:24-32. Insome embodiments, the heterologous CBM comprises, consists essentiallyof, or consists of a CBM amino acid sequence that has at least about 80%to about 95% amino acid sequence similarity or identity with the aminoacid sequence of any one of SEQ ID NOs:24-32. In some embodiments,heterologous CBM comprises, consists essentially of, consists of a CBMamino acid sequence that has at least about 95% amino acid sequencesimilarity or identity (e.g., 95%, 96%, 97%, 98%, 99%, or more) with theamino acid sequence of any one of SEQ ID NOs:24-32.

In some embodiments, the heterologous CBM comprises a metal bindingsite. In some embodiments, the metal binding site of the heterologousCBM binds calcium and/or magnesium. A metal binding site on a CBM may becoordinated by charged atoms and may bind in and into loop motifs on asurface of the CBM and/or the protein that the CBM in present in. Insome embodiments, the metal may be involved in the binding of acarbohydrate. In some embodiments, the metal may not be relevant to thebinding of the carbohydrate function of the CBM in the protein in whichthe CBM is naturally located.

In some embodiments, the modified Vip3 polypeptide is pesticidalagainst, for example, insects. Accordingly, in some embodiments, themodified Vip3 polypeptide is pesticidal against an insect, for example,a lepidopteran insect.

Accordingly, in some embodiments, insect pests include withoutlimitation insects selected from the orders Coleoptera, Diptera,Hymenoptera, Lepidoptera, Mallophaga, Homoptera, Hemiptera, Orthroptera,Thysanoptera, Dermaptera, Isoptera, Anoplura, Siphonaptera, Trichoptera,and the like. In some embodiments, insect pests include withoutlimitation Ostrinia nubilalis (European corn borer), Plutella xylostella(diamondback moth), Spodoptera frugiperda (fall armyworm), Agrotisipsilon (black cutworm), Agrotis orthogonia (pale western cutworm),Striacosta albicosta (western bean cutworm), Helicoverpa zea (cornearworm), Heliothis virescens (tobacco budworm), Spodoptera exigua (beetarmyworm), Helicoverpa punctigera (native budworm), Helicoverpa armigera(cotton bollworm), Manduca sexta (tobacco hornworm), Trichoplusia ni(cabbage looper), Pectinophora gossypiella (pink bollworm), Diatraeagrandiosella (southwestern corn borer), Diatraea saccharalis (sugarcaneborer), Elasmopalpus lignosellus (lesser cornstalk borer), Psuedoplusiaincludens (soybean looper), Anticarsia gemmatalis (velvetbeancaterpillar), Plathypena scabra (green cloverworm), Homoeosomaelectellum (sunflower head moth), Cochylis hospes (banded sunflowermoth), or any combination thereof.

In some embodiments, a composition comprising a modified Vip3polypeptide is provided. In some embodiments, the composition comprisingthe modified Vip3 polypeptide may be prepared from an extract of atransgenic plant or plant part (e.g., seed), said transgenic plant orplant part comprising a nucleotide sequence encoding said modified Vip3polypeptide. In some embodiments, the composition comprising themodified Vip3 polypeptide may be produced by expressing a polynucleotideencoding a polypeptide of the invention in bacterial cells capable ofexpressing the polynucleotide. In some embodiments, the compositioncomprises the modified Vip3 polypeptide in an agriculturally acceptablecarrier.

As used herein an “agriculturally-acceptable carrier” may includenatural or synthetic, organic or inorganic material, which may becombined with the active component to facilitate its application to theplant, or part thereof. An agriculturally-acceptable carrier includes,but is not limited to, inert components, dispersants, surfactants,adjuvants, tackifiers, stickers, binders, or combinations thereof, thatmay be used in agricultural formulations. Another agriculturallyacceptable carrier may be a transgenic plant or plant part.

Such compositions may be applied in any manner that brings thepesticidal polypeptides in contact with the pests, resulting in toxiceffect and control of the pest(s). Accordingly, the compositions may beapplied to the surfaces of plants or plant parts, including seeds,leaves, flowers, stems, tubers, roots, and the like. Thus, thecomposition(s) of the modified Vip3 polypeptides may be delivered inmany recognized ways, e.g., orally by ingestion by the pest or bycontact with the pest via transgenic plant expression, formulatedprotein composition(s), sprayable protein composition(s), a bait matrix,or any other art-recognized toxin delivery system.

In some embodiments, the invention provides a nucleic acid moleculecomprising, consisting essentially of, consisting of a nucleotidesequence encoding one or more than one of the modified Vip3 polypeptidesdescribed herein. In some embodiments, the nucleic acid moleculecomprises, consists essentially of, or consists of one or more than oneof the nucleotide sequence(s) of SEQ ID NOs:7-23 and/or SEQ IDNOs:33-56. In some embodiments, a nucleotide sequence encoding amodified Vip3 polypeptide may be codon optimized for expression in aparticular host organism or host cell.

Thus, in some embodiments, a nucleic acid of this invention is expressedin transgenic plants. For expression in transgenic plants, thenucleotide sequences encoding the modified Vip3 polypeptides of theinvention may require other modifications and/or optimization. Althoughin many cases, nucleotide sequences from microbial organisms can beexpressed in plants at high levels without modification, low expressionin transgenic plants may result from microbial nucleotide sequenceshaving codons that are not preferred in plants. It is known in the artthat all organisms have specific preferences for codon usage, and thecodons of the nucleic acids/nucleotide sequences described herein can bechanged to conform with plant preferences, while maintaining the aminoacid sequence encoded thereby. Furthermore, high expression in plants isbest achieved from coding sequences that have at least about 35% GCcontent, preferably more than about 45%, more preferably more than about50%, and most preferably more than about 60%. Microbial nucleic acidsthat have low GC contents may express poorly in plants due to theexistence of ATTTA motifs that may destabilize messages, and AATAAAmotifs that may cause inappropriate polyadenylation. Although nucleotidesequences may be adequately expressed in both monocotyledonous anddicotyledonous plant species, sequences can be modified to account forthe specific codon preferences and GC content preferences ofmonocotyledons or dicotyledons as these preferences have been shown todiffer (Murray et al. Nucl. Acids Res. 17:477-498 (1989)). In addition,the nucleotide sequences can be screened for the existence ofillegitimate splice sites that may cause message truncation. All changesrequired to be made within the nucleic acids/nucleotide sequences suchas those described above are made using well known techniques of sitedirected mutagenesis, PCR, and synthetic gene construction (See, e.g.,EP 0 385 962, EP 0 359 472, and WO 93/07278.

In some embodiments, the invention provides transgenic non-human hostcells comprising the nucleic acid molecules of the invention. Anon-human host cell can include, but is not limited to, a plant cell, abacterial cell, a fungal (e.g., yeast) cell, or an insect cell. In someembodiments, the transgenic host cell is a transgenic plant cell or atransgenic bacterial cell. In some embodiments, the plant cell is anon-propagating cell.

In some embodiments, the invention provides a plant, plant part and/orplant cell comprising the nucleic acid molecules of the invention.Non-limiting examples of plants useful with this invention includevegetable crops, including artichokes, kohlrabi, arugula, leeks,asparagus, lettuce (e.g., head, leaf, romaine), bok choy, malanga,melons (e.g., muskmelon, watermelon, crenshaw, honeydew, cantaloupe),cole crops (e.g., brussels sprouts, cabbage, cauliflower, broccoli,collards, kale, chinese cabbage, bok choy) cardoni, carrots, napa, okra,onions, celery, parsley, chick peas, parsnips, chicory, peppers,potatoes, cucurbits (e.g., marrow, cucumber, zucchini, squash, pumpkin),radishes, dry bulb onions, rutabaga, eggplant (also called brinjal),salsify, escarole, shallots, endive, garlic, spinach, green onions,squash, greens, beet (sugar beet and fodder beet), sweet potatoes, swisschard, horseradish, tomatoes, turnips, and spices; a fruit and/or vinecrop such as apples, apricots, cherries, nectarines, peaches, pears,plums, prunes, cherry, quince, almonds, chestnuts, filberts, pecans,pistachios, walnuts, citrus, blueberries, boysenberries, cranberries,currants, loganberries, raspberries, strawberries, blackberries, grapes,avocados, bananas, kiwi, persimmons, pomegranate, pineapple, tropicalfruits, pomes, melon, mango, papaya, and lychee; a field crop plant suchas clover, alfalfa, evening primrose, meadow foam, corn/maize (field,sweet, popcorn), hops, jojoba, peanuts, rice, safflower, small grains(barley, oats, rye, wheat, etc.), sorghum, tobacco, kapok, a leguminousplant (beans, lentils, peas, soybeans), an oil plant (rape, mustard,poppy, olive, sunflower, coconut, castor oil plant, cocoa bean,groundnut), Arabidopsis, grasses (turf grasses, ornamental grasses), afibre plant (cotton, flax, hemp, jute), lauraceae (cinnamon, camphor),or a plant such as coffee, sugar cane, tea, and natural rubber plants;and/or a bedding plant such as a flowering plant, a cactus, a succulentand/or an ornamental plant, as well as trees such as forest(broad-leaved trees and evergreens, such as conifers), fruit,ornamental, and nut-bearing trees, as well as shrubs and other nurserystock.

In particular embodiments, a plant, plant part or plant cell of thisinvention may be sorghum, wheat, sunflower, tomato, a cole crop, cotton,rice, soybean, sugar beet, sugar cane, tobacco, barley, oilseed rapeand/or maize. In some embodiments, the plant is maize. In someembodiments, the plant is soybean.

In representative embodiments, a plant comprising the nucleic acidmolecules of the invention and expressing the Vip3 polypeptide is maizeand the Vip3 polypeptide produced by the plant is pestidical against aninsect pest from the order Coleoptera, Diptera, Hymenoptera,Lepidoptera, Mallophaga, Homoptera, Hemiptera, Orthroptera,Thysanoptera, Dermaptera, Isoptera, Anoplura, Siphonaptera, Trichoptera,or any combination thereof. In some embodiments, a plant comprising thenucleic acid molecules of the invention and expressing the Vip3polypeptide is maize and the Vip3 polypeptide produced by the plant ispestidical against an insect pest from the order Lepidoptera. In someembodiments, a plant comprising the nucleic acid molecules of theinvention and expressing the Vip3 polypeptide is maize and the Vip3polypeptide produced by the plant is pestidical against, for example,Ostrinia nubilalis (European corn borer), Spodoptera frugiperda (fallarmyworm), Agrotis ipsilon (black cutworm), Agrotis orthogonia (palewestern cutworm), Striacosta albicosta (western bean cutworm),Helicoverpa zea (corn earworm), Spodoptera exigua (beet armyworm),Helicoverpa punctigera (native budworm), Helicoverpa armigera (cottonbollworm), Diatraea grandiosella (southwestern corn borer), Diatraeasaccharalis (sugarcane borer), Elasmopalpus lignosellus (lessercornstalk borer), or any combination thereof. In some embodiments, aplant comprising the nucleic acid molecules of the invention andexpressing the Vip3 polypeptide is maize and the Vip3 polypeptideproduced by the plant is pestidical against Ostrinia nubilalis (Europeancorn borer), Spodoptera frugiperda (fall armyworm), Helicoverpa zea(corn earworm), or Agrotis ipsilon (black cutworm) or any combinationthereof.

In representative embodiments, a plant comprising the nucleic acidmolecules of the invention and expressing the Vip3 polypeptide issoybean and the Vip3 polypeptide produced by the plant is pestidicalagainst an insect pest from the order Coleoptera, Diptera, Hymenoptera,Lepidoptera, Mallophaga, Homoptera, Hemiptera, Orthroptera,Thysanoptera, Dermaptera, Isoptera, Anoplura, Siphonaptera, Trichoptera,or any combination thereof. In some embodiments, a plant comprising thenucleic acid molecules of the invention and expressing the Vip3polypeptide is soybean and the Vip3 polypeptide produced by the plant ispestidical against an insect pest from the order Lepidoptera. In someembodiments, a plant comprising the nucleic acid molecules of theinvention and expressing the Vip3 polypeptide is soybean and the Vip3polypeptide produced by the plant is pestidical against, for example,Spodoptera frugiperda (fall armyworm), Agrotis ipsilon (black cutworm),Agrotis orthogonia (pale western cutworm), Helicoverpa zea (cornearworm), Heliothis virescens (tobacco budworm), Spodoptera exigua (beetarmyworm), Helicoverpa punctigera (native budworm), Helicoverpa armigera(cotton bollworm), Trichoplusia ni (cabbage looper), Elasmopalpuslignosellus (lesser cornstalk borer), Psuedoplusia includens (soybeanlooper), Anticarsia gemmatalis (velvetbean caterpillar), Plathypenascabra (green cloverworm), Homoeosoma electellum (sunflower head moth),or Cochylis hospes (banded sunflower moth), or any combination thereof.In some embodiments, a plant comprising the nucleic acid molecules ofthe invention and expressing the Vip3 polypeptide is soybean and theVip3 polypeptide produced by the plant is pestidical against Ostrinianubilalis (European corn borer), Spodoptera frugiperda (fall armyworm),Helicoverpa zea (corn earworm), or Agrotis ipsilon (black cutworm) orany combination thereof.

In some embodiments, a virus, such as a baculovirus, may comprise apolynucleotide encoding a modified Vip3 polypeptide of this invention inits genome. Said recombinant viruses can express large amounts of thecorresponding modified Vip3 polypeptide after infection of eukaryoticcells suitable for virus replication and expression of thepolynucleotide. The pesticidal polypeptide thus produced can be used asa pesticidal or insecticidal agent. Alternatively, viruses (e.g.,baculoviruses) engineered to include one or more polynucleotides of thisinvention can be used to infect insects in vivo and kill them either byexpression of the pesticidal polypeptide or by a combination of viralinfection and expression of the pesticidal polypeptide.

Bacterial cells can also be hosts for the expression of the nucleicacids of the invention. In one embodiment, non-pathogenic symbioticbacteria, which are able to live and replicate within plant tissues,so-called endophytes, or non-pathogenic symbiotic bacteria, which arecapable of colonizing the phyllosphere or the rhizosphere, so-calledepiphytes, are used. Such bacteria include bacteria of the generaAgrobacterium, Alcaligenes, Azospirillum, Azotobacter, Bacillus,Clavibacter, Enterobacter, Erwinia, Flavobacter, Klebsiella,Pseudomonas, Rhizobium, Serratia, Streptomyces and Xanthomonas.Symbiotic fungi, such as Trichoderma and Gliocladium are also possiblehosts for expression of the inventive nucleic acids for the samepurpose.

Thus, in some embodiments, as biological pesticidal control agents, thepesticidal polypeptides of the invention may be produced by expressionof the polynucleotides encoding the polypeptides of the invention inheterologous host cells capable of expressing the polynucleotides.Accordingly, in some embodiments, a yeast, bacterial or plant cellcomprising one or more polynucleotides of the invention is provided.

Techniques for the transformation of the various organisms/host cellsare known in the art. For example, the expression vectors pKK223-3 andpKK223-2 may be used to express heterologous genes in E. coli, either intranscriptional or translational fusion, behind a tac or trc promoter.For the expression of operons encoding multiple ORFs, one procedure isto insert the operon into a vector such as pKK223-3 in transcriptionalfusion, allowing the cognate ribosome binding site of the heterologousgenes to be used. Techniques for overexpression in gram-positive speciessuch as Bacillus are also known in the art and may be used in thecontext of this invention (Quax et al. In: Industrial Microorganisms:Basic and Applied Molecular Genetics, Eds. Baltz et al., AmericanSociety for Microbiology, Washington (1993)). Alternate systems foroverexpression rely, for example, on yeast vectors and include the useof Pichia, Saccharomyces and/or Kluyveromyces (Sreekrishna, In:Industrial Microorganisms: Basic and Applied Molecular Genetics, Baltz,Hegeman, and Skatrud eds., American Society for Microbiology, Washington(1993); Dequin & Barre, Biotechnology L2:173-177 (1994); van den Berg etal., Biotechnology 8:135-139 (1990)).

Procedures for transforming plants are well known and routine in the artand are described throughout the literature. Non-limiting examples ofmethods for transformation of plants include transformation viabacterial-mediated nucleic acid delivery (e.g., via Agrobacteria),viral-mediated nucleic acid delivery, silicon carbide or nucleic acidwhisker-mediated nucleic acid delivery, liposome mediated nucleic aciddelivery, microinjection, microparticle bombardment,calcium-phosphate-mediated transformation, cyclodextrin-mediatedtransformation, electroporation, nanoparticle-mediated transformation,sonication, infiltration, PEG-mediated nucleic acid uptake, as well asany other electrical, chemical, physical (mechanical) and/or biologicalmechanism that results in the introduction of nucleic acid into theplant cell, including any combination thereof. General guides to variousplant transformation methods known in the art include Miki et al.(“Procedures for Introducing Foreign DNA into Plants” in Methods inPlant Molecular Biology and Biotechnology, Glick, B. R. and Thompson, J.E., Eds. (CRC Press, Inc., Boca Raton, 1993), pages 67-88) andRakowoczy-Trojanowska (Cell. Mol. Biol. Lett. 7:849-858 (2002)).

For Agrobacterium-mediated transformation, binary vectors or vectorscarrying at least one T-DNA border sequence are suitable, whereas fordirect gene transfer (e.g., particle bombardment and the like) anyvector is suitable and linear DNA containing only the construction ofinterest may be used. In the case of direct gene transfer,transformation with a single DNA species or co-transformation may beused (Schocher et al., Biotechnology 4:1093-1096 (1986)). For bothdirect gene transfer and Agrobacterium-mediated transfer, transformationis usually (but not necessarily) undertaken with a selectable markerthat may be a positive selection (Phosphomannose Isomerase), provideresistance to an antibiotic (kanamycin, hygromycin or methotrexate) or aherbicide (glyphosate or basta). However, the choice of selectablemarker is not critical to the invention.

Agrobacterium-mediated transformation is a commonly used method fortransforming plants, in particular, dicot plants, because of its highefficiency of transformation and because of its broad utility with manydifferent species. Agrobacterium-mediated transformation typicallyinvolves transfer of the binary vector carrying the foreign DNA ofinterest to an appropriate Agrobacterium strain that may depend on thecomplement of vir genes carried by the host Agrobacterium strain eitheron a co-resident Ti plasmid or chromosomally (Uknes et al. (1993) PlantCell 5:159-169). The transfer of the recombinant binary vector toAgrobacterium can be accomplished by a triparental mating procedureusing Escherichia coli carrying the recombinant binary vector, a helperE. coli strain that carries a plasmid that is able to mobilize therecombinant binary vector to the target Agrobacterium strain.Alternatively, the recombinant binary vector can be transferred toAgrobacterium by nucleic acid transformation (Höfgen & Willmitzer (1988)Nucleic Acids Res. 16:9877).

Transformation of a plant by recombinant Agrobacterium usually involvesco-cultivation of the Agrobacterium with explants from the plant andfollows methods well known in the art. Transformed tissue is regeneratedon selection medium carrying an antibiotic or herbicide resistancemarker between the binary plasmid T-DNA borders.

As discussed previously, another method for transforming plants, plantparts and plant cells involves propelling inert or biologically activeparticles at plant tissues and cells. See, e.g., U.S. Pat. Nos.4,945,050; 5,036,006 and 5,100,792. Generally, this method involvespropelling inert or biologically active particles at the plant cellsunder conditions effective to penetrate the outer surface of the celland afford incorporation within the interior thereof. When inertparticles are utilized, the vector can be introduced into the cell bycoating the particles with the vector containing the nucleic acid ofinterest. Alternatively, a cell or cells can be surrounded by the vectorso that the vector is carried into the cell by the wake of the particle.Biologically active particles (e.g., a dried yeast cell, a driedbacterium or a bacteriophage, each containing one or more nucleic acidssought to be introduced) also can be propelled into plant tissue.

In some embodiments, a polynucleotide of the invention may be directlytransformed into the plastid genome. A major advantage of plastidtransformation is that plastids are generally capable of expressingbacterial genes without substantial modification, and plastids arecapable of expressing multiple open reading frames under control of asingle promoter. Plastid transformation technology is extensivelydescribed in U.S. Pat. Nos. 5,451,513, 5,545,817, and 5,545,818, in PCTapplication no. WO 95/16783, and in McBride et al. (1994) Proc. Nati.Acad. Sci. USA 91, 7301-7305. The basic technique for chloroplasttransformation involves introducing regions of cloned plastid DNAflanking a selectable marker together with the gene of interest into asuitable target tissue, e.g., using biolistics or protoplasttransformation (e.g., calcium chloride or PEG mediated transformation).The 1 to 1.5 kb flanking regions, termed targeting sequences, facilitatehomologous recombination with the plastid genome and thus allow thereplacement or modification of specific regions of the plastome.Initially, point mutations in the chloroplast 16S rRNA and rps12 genesconferring resistance to spectinomycin and/or streptomycin can beutilized as selectable markers for transformation (Svab, Z.,Hajdukiewicz, P., and Maliga, P. (1990) Proc. Natl. Acad. Sci. USA 87,8526-8530; Staub, J. M., and Maliga, P. (1992) Plant Cell 4, 39-45). Thepresence of cloning sites between these markers allows creation of aplastid targeting vector for introduction of foreign genes (Staub, J.M., and Maliga, P. (1993) EMBO J. 12, 601-606). Substantial increases intransformation frequency can be obtained by replacement of the recessiverRNA or r-protein antibiotic resistance genes with a dominant selectablemarker, the bacterial aadA gene encoding the spectinomycin-cletoxifyingenzyme aminoglycoside-3′-adenyltransf erase (Svab, Z., and Maliga, P.(1993) Proc. Natl. Acad. Sci. USA 90, 913-917). Previously, this markerhad been used successfully for high-frequency transformation of theplastid genome of the green alga Chlamydomonas reinhardtii(Goldschmidt-Clermont, M. (1991) Nucl. Acids Res. 19:4083-4089). Otherselectable markers useful for plastid transformation are known in theart and encompassed within the scope of the invention. Typically,approximately 15-20 cell division cycles following transformation arerequired to reach a homoplastidic state. Plastid expression, in whichgenes are inserted by homologous recombination into all of the severalthousand copies of the circular plastid genome present in each plantcell, takes advantage of the enormous copy number advantage overnuclear-expressed genes to permit expression levels that can readilyexceed 10% of the total soluble plant protein. In one embodiment, apolynucleotide of the invention can be inserted into a plastid-targetingvector and transformed into the plastid genome of a desired plant host.Thus, plants homoplastic for plastid genomes containing a nucleotidesequence of the invention can be obtained, which are capable of highexpression of the polynucleotide.

Methods of selecting for transformed, transgenic plants, plant cellsand/or plant tissue culture are routine in the art and can be employedin the methods of the invention provided herein.

A polynucleotide therefore can be introduced into the plant, plant partand/or plant cell in any number of ways that are well known in the art,as described above. Therefore, no particular method for introducing oneor more polynucleotides into a plant is relied upon, rather any methodthat allows the one or more polynucleotides to gain access to theinterior of at least one cell of the plant may be used. Where more thanone polynucleotides is to be introduced, the respective polynucleotidesmay be assembled as part of a single nucleic acid molecule, or asseparate nucleic acid molecules, and may be located on the same ordifferent nucleic acid molecules. Accordingly, the polynucleotides maybe introduced into the cell of interest in a single transformationevent, in separate transformation events, or, for example, in plants, aspart of a breeding protocol.

In some embodiments of this invention, the introduced nucleic acidmolecule may be maintained in the plant cell stably if it isincorporated into a non-chromosomal autonomous replicon or integratedinto the plant chromosome(s). Alternatively, the introduced nucleic acidmolecule may be present on an extra-chromosomal non-replicating vectorand be transiently expressed or transiently active. Whether present inan extra-chromosomal non-replicating vector or a vector that isintegrated into a chromosome, the nucleic acid molecule may be presentin a plant expression cassette. A plant expression cassette may containregulatory sequences that drive gene expression in plant cells that areoperatively linked so that each sequence can fulfill its function, forexample, termination of transcription by polyadenylation signals.Exemplary polyadenylation signals can be those originating fromAgrobacterium tumefaciens T-DNA such as the gene known as octopinesynthase of the Ti-plasmid pTiACH5 (Gielen et al. EMBO J. 3:835 (1984))or functional equivalents thereof, but also all other terminatorsfunctionally active in plants are suitable. A plant expression cassetteof this invention may also contain other operatively linked sequenceslike translational enhancers such as the overdrive-sequence containingthe 5′-untranslated leader sequence from tobacco mosaic virus enhancingthe polypeptide per RNA ratio (Gallie et al. Nucl. Acids Research15:8693-8711 (1987)).

Further, as is well known in the art, intact transgenic plants may beregenerated from transformed plant cells, plant tissue culture and/orcultured protoplasts using any of a variety of known techniques. Plantregeneration from plant cells, plant tissue culture and/or culturedprotoplasts is described, for example, in Evans et al. (Handbook ofPlant Cell Cultures, Vol. 1, MacMilan Publishing Co. New York (1983));and Vasil I. R. (ed.) (Cell Culture and Somatic Cell Genetics of Plants,Acad. Press, Orlando, Vol. I (1984), and Vol. II (1986)).

Additionally, the genetic properties engineered into the transgenicseeds and plants, plant parts, and/or plant cells of the inventiondescribed above can be passed on by sexual reproduction or vegetativegrowth and therefore can be maintained and propagated in progeny plants.Generally, maintenance and propagation make use of known agriculturalmethods developed to fit specific purposes such as harvesting, sowing ortilling.

Accordingly, the present invention provides transgenic plants, plantparts and plant cells and plants regenerated therefrom comprising anucleic acid molecule comprising, consisting essentially of, orconsisting of a nucleotide sequence encoding a modified Vip3 polypeptideof the invention. In some embodiments, the transgenic plant, plant partor plant cell is a selected from the group of plants consisting ofsorghum, wheat, sunflower, tomato, cole crop, cotton, rice, soybean,sugar beet, sugar cane, tobacco, barley, oilseed rape and maize.

In some embodiments of the invention a harvested product produced fromthe transgenic cells, plants and/or plant parts of the invention may beprovided, as well as a processed product produced from said harvestedproduct. A harvested product may be a whole plant or any plant part, asdescribed herein, wherein said harvested product comprises a nucleicacid molecule/nucleotide sequence or polypeptide of the invention. Thus,in some embodiments, a non-limiting example of a harvested productincludes a seed, a fruit, a flower or part thereof (e.g., an anther, astigma, and the like), a leaf, a stem, a stalk, a root, a tuber, and thelike. In particular embodiments, the harvested product is a seed,wherein the seed comprised the nucleic acid molecule(s)/nucleotidesequence(s) and/or polypeptide(s) of the invention in its genome.

In some embodiments, a processed product includes, but is not limitedto, a flour, meal, oil, starch, sugar, fiber, biofuel or cereal, and thelike, or a product produced therefrom. In some embodiments the processedproduct is an extract from the seed, wherein the extract comprises anucleic acid molecule, nucleotide sequence or polypeptide of theinvention. In some embodiments, the processed product comprises anucleic acid molecule/nucleotide sequence or polypeptide of theinvention.

In some embodiments, the invention further provides a crop comprising aplurality of the plants of the invention planted together in anagricultural field. In some embodiments, the invention provides a cropcomprising a plurality of the transgenic plants of the invention plantedtogether in a golf course, a residential lawn, a road side, an athleticfield, and/or a recreational field.

Also provided herein are methods of using the modified Vip3 polypeptidesof the invention, and/or the nucleic acid molecules/polynucleotidesencoding said modified Vip3 polypeptides.

Accordingly, in some embodiments, a method of producing a plant, plantpart or plant cell with increased resistant or tolerance to one or morepests is provided, the method comprising introducing one or more nucleicacid molecules comprising one or more nucleotide sequences encoding oneor more modified Vip3 polypeptides of the invention into a plant, plantpart or plant cell to produce a transgenic plant, plant part or plantcell that expresses the one or more nucleic acid molecules, therebyexpressing the one or more modified Vip3 polypeptides and increasingresistance or tolerance to one or more pests in said transgenic plant,plant part or plant cell as compared with a control plant, plant part orplant cell that does not comprise said one or more nucleic acidmolecules. In some embodiments, the method comprises regenerating atransgenic plant from said transgenic plant cell or plant part, whereinthe transgenic plant comprises in its genome the one or more nucleicacid molecules and has increased pesticidal activity.

In another aspect, a method of increasing pesticidal activity in aplant, plant part or plant cell is provided, the method comprisingintroducing one or more nucleic acid molecules comprising one or morenucleotide sequences encoding one or more modified Vip3 polypeptides ofthe invention into a plant, plant part or plant cell to produce atransgenic plant, plant part or plant cell that expresses the one ormore nucleic acid molecules, thereby expressing (producing) the one ormore modified Vip3 polypeptides and increasing pesticidal activity inthe transgenic plant, plant part or plant cell as compared with acontrol plant, plant part or plant cell that does not comprise said oneor more nucleic acid molecules. In some embodiments, the methodcomprises regenerating a transgenic plant from said transgenic plantcell or plant part, wherein the transgenic plant comprises in its genomethe one or more nucleic acid molecules and has increased pesticidalactivity.

In some embodiments, a method of providing a farmer with a means ofcontrolling a plant pest is provided, the method comprising supplying tothe farmer plant material or bacteria, said plant material or bacteriacomprising a nucleic acid molecule that encodes the modified Vip3polypeptide of the invention.

In some embodiments, a method of producing the modified Vip3 polypeptideof the invention is provided, the method comprising the steps of: (a)transforming a host cell with a recombinant nucleic acid moleculecomprising a nucleotide sequence encoding for the modified Vip3polypeptide; and (b) culturing the host cell of step (a) underconditions in which the host cell expresses the recombinant nucleic acidmolecule, thereby producing the modified Vip3 polypeptide.

In some embodiments of the invention, a method of producing a modifiedVip3 polypeptide is provided, the method comprising, growing a host cellof the invention under conditions which allow expression of the modifiedVip3 polypeptide; and recovering the modified Vip3 polypeptide.

In some embodiments, a method of reducing damage in a transgenic plantcaused by a plant pest, the method comprising planting a transgenicplant seed comprising a nucleic acid molecule that expresses themodified Vip3 polypeptide or the invention, thereby reducing damagecaused by the pest to a transgenic plant grown from the transgenic plantseed. In some embodiments, the nucleic acid molecule(s) is/are comprisedin an expression cassette or a recombinant vector.

In any of the embodiments described herein, the nucleic acid moleculesof the invention may be comprised in one or more expression cassette(s)and/or vector(s), wherein said nucleic acid molecules may be in operableassociation with one or more promoters (and/or other regulatoryelements) that function in the host cell (e.g., plant, bacteria, etc).In some embodiments, the one or more expression cassette(s) or vector(s)may comprise a selectable marker. In some embodiments, the one or moreexpression cassette(s) or vector(s) do not comprise a selectable marker.

In some embodiments, the invention provides a method of controllingpests comprising, contacting the pests with a pesticidally effectiveamount of a composition of the invention. In some embodiments of theinvention, a method of protecting a plant and/or a plant propagationmaterial is provided, the method comprising contacting the plant and/orplant propagation material with a pesticidally effective amount of acomposition of the invention.

The modified Vip3 polypeptides of the invention may be used incombination with other pesticidal principles or crop protection products(i.e., pestidical active ingredients) to increase pest target range.Thus, the modified Vip3 polypeptide may be used in combination withother pesticidal principles of a distinct nature for the preventionand/or management of insect resistance. Other insecticidal principlesinclude, for example, protease inhibitors (both serine and cysteinetypes), lectins, alpha-amylase, peroxidase and cholesterol oxidase.Similarly, Cry proteins, such as the proteins in the CryIA, CryIB andCryIC families, are also useful in combination with the modified Vip3proteins of the present invention.

The co-expression of more than one insecticidal principle in the sametransgenic plant or plant part (e.g., seed) can be achieved bygenetically engineering a plant to contain and express all the genesnecessary. Alternatively, a plant, Parent 1, can be geneticallyengineered for the expression of genes of the present invention. Asecond plant, Parent 2, can be genetically engineered for the expressionof a supplemental insect control principle. By crossing Parent 1 withParent 2, progeny plants are obtained which express all the genesintroduced into Parents 1 and 2.

Transgenic plants or plant parts or transgenic seed of the invention mayalso be treated with a pesticidal active ingredient/crop protectionproduct. Where both the pesticidal active ingredient and the transgenicplant or plant part or transgenic seed of the invention are activeagainst the same target insect, the combination is useful (i) in amethod for enhancing activity of a modified Vip3 polypeptide of theinvention against the target insect and (ii) in a method for preventingdevelopment of resistance to modified Vip3 polypeptide of the inventionby providing a second mechanism of action against the target insect.Thus, the invention provides a method of enhancing activity against orpreventing development of resistance in a target insect, comprising, forexample, applying an insecticidal seed coating as described in U.S. Pat.Nos. 5,849,320 and 5,876,739, herein incorporated by reference to atransgenic seed comprising one or more modified Vip3 polypeptide of theinvention.

Therefore in one embodiment, the invention encompasses a method ofcontrolling crop pests by providing a transgenic plant, or transgenicplant part (e.g., transgenic seed) of the invention and applying to thetransgenic plant, plant part or seed a pesticidal active ingredient/cropprotection product. In doing so, the activity of a modified Vip3polypeptide of the invention against a target insect is enhanced and(ii) in a method for preventing development of resistance to modifiedVip3 polypeptide of the invention by providing a second mechanism ofaction against the target insect. Further, the application of a cropprotection product to a transgenic plant or plant part may be done toincrease the number of crop pests controlled by applying a cropprotection that is effective against additional crop pests.

Such active ingredients that may be applied to a transgenic plant and/ora transgenic plant part (e.g., seed) of the invention as described aboveincludes, without limitation, (1) Acetylcholine esterase (AChE)inhibitors, for example carbamates, for example alanycarb, aldicarb,aldoxycarb, allyxycarb, aminocarb, bendiocarb, benfuracarb, bufencarb,butacarb, butocarboxim, butoxycarboxim, carbaryl, carbofuran,carbosulfan, cloethocarb, dimetilan, ethiofencarb, fenobucarb,fenothiocarb, formetanate, furathiocarb, isoprocarb, metam-sodium,methiocarb, methomyl, metolcarb, oxamyl, pirimicarb, promecarb,propoxur, thiodicarb, thiofanox, trimethacarb, XMC and xylylcarb; ororganophosphates, for example acephate, azamethiphos, azinphos (-methyl,-ethyl), bromophos-ethyl, bromfenvinfos (-methyl), butathiofos,cadusafos, carbophenothion, chlorethoxyfos, chlorfenvinphos,chlormephos, chlorpyrifos (-methyl/-ethyl), coumaphos, cyanofenphos,cyanophos, chlorfenvinphos, demeton-5-methyl, demeton-5-methylsulphone,dialifos, diazinon, dichlofenthion, dichlorvos/DDVP, dicrotophos,dimethoate, dimethylvinphos, dioxabenzofos, disulphoton, EPN, ethion,ethoprophos, etrimfos, famphur, fenamiphos, fenitrothion,fensulphothion, fenthion, flupyrazofos, fonofos, formothion,fosmethilan, fosthiazate, heptenophos, iodofenphos, iprobenfos,isazofos, isofenphos, isopropyl O-salicylate, isoxathion, malathion,mecarbam, methacrifos, methamidophos, methidathion, mevinphos,monocrotophos, naled, omethoate, oxydemeton-methyl, parathion(-methyl/-ethyl), phenthoate, phorate, phosalone, phosmet, phosphamidon,phosphocarb, phoxim, pirimiphos (-methyl/-ethyl), profenofos, propaphos,propetamphos, prothiofos, prothoate, pyraclofos, pyridaphenthion,pyridathion, quinalphos, sebufos, sulphotep, sulprofos, tebupirimfos,temephos, terbufos, tetrachlorvinphos, thiometon, triazophos,trichlorfon, vamidothion, and imicyafos. (2) GABA-gated chloride channelantagonists, for example organochlorines, for example camphechlor,chlordane, endosulfan, gamma-HCH, HCH, heptachlor, lindane andmethoxychlor; or fiproles (phenylpyrazoles), for example acetoprole,ethiprole, fipronil, pyrafluprole, pyriprole, vaniliprole. (3)Sodium-channel modulators/voltage-dependent sodium channel blockers, forexample pyrethroids, for example acrinathrin, allethrin (d-cis-trans,d-trans), beta-cyfluthrin, bifenthrin, bioallethrin,bioallethrin-S-cyclopentyl isomer, bioethanomethrin, biopermethrin,bioresmethrin, chlovaporthrin, cis-cypermethrin, cis-resmethrin,cis-permethrin, clocythrin, cycloprothrin, cyfluthrin, cyhalothrin,cypermethrin (alpha-, beta-, theta-, zeta-), cyphenothrin, deltamethrin,empenthrin (1R isomer), esfenvalerate, etofenprox, fenfluthrin,fenpropathrin, fenpyrithrin, fenvalerate, flubrocythrinate,flucythrinate, flufenprox, flumethrin, fluvalinate, fubfenprox,gamma-cyhalothrin, imiprothrin, kadethrin, lambda-cyhalothrin,metofluthrin, permethrin (cis-, trans-), phenothrin (1R-trans isomer),prallethrin, profluthrin, protrifenbute, pyresmethrin, resmethrin, RU15525, silafluofen, tau-fluvalinate, tefluthrin, terallethrin,tetramethrin (1R isomer), tralomethrin, transfluthrin, ZXI 8901,pyrethrin (pyrethrum), eflusilanat; DDT; or methoxychlor. (4)Nicotinergic acetylcholine receptor agonists/antagonists, for exampleChloronicotinyls, for example acetamiprid, clothianidin, dinotefuran,imidacloprid, imidaclothiz, nitenpyram, nithiazine, thiamethoxam,AKD-1022, nicotine, bensultap, cartap, thiosultap-sodium, and thiocylam.(5) Allosteric acetylcholine receptor modulators (agonists), for examplespinosyns, for example spinosad and spinetoram. (6) Chloride channelactivators, for example mectins/macrolides, for example abamectin,emamectin, emamectin benzoate, ivermectin, lepimectin, and milbemectin;or juvenile hormone analogues, for example hydroprene, kinoprene,methoprene, epofenonane, triprene, fenoxycarb, pyriproxifen, anddiofenolan. (7) Active ingredients with unknown or nonspecificmechanisms of action, for example fumigants, for example methyl bromide,chloropicrin and sulphuryl fluoride; selective antifeedants, for examplecryolite, pymetrozine, pyrifluquinazon and flonicamid; or mite growthinhibitors, for example clofentezine, hexythiazox, etoxazole. (8)Inhibitors of oxidative phosphorylation, ATP disruptors, for examplediafenthiuron; organotin compounds, for example azocyclotin, cyhexatinand fenbutatin oxide; or propargite, tetradifon. (9) Oxidativephosphorylation decouplers which interrupt the H-proton gradient, forexample chlorfenapyr, binapacyrl, dinobuton, dinocap and DNOC. (10)Microbial disruptors of the insect gut membrane, for example Bacillusthuringiensis strains. (11) Chitin biosynthesis inhibitors, for examplebenzoylureas, for example bistrifluoron, chlorfluazuron, diflubenzuron,fluazuron, flucycloxuron, flufenoxuron, hexaflumuron, lufenuron,novaluron, noviflumuron, penfluoron, teflubenzuron or triflumuron. (12)Buprofezin. (13) Moulting disruptors, for example cyromazine. (14)Ecdysone agonists/disruptors, for example diacylhydrazines, for examplechromafenozide, halofenozide, methoxyfenozide, tebufenozide, andfufenozide (JS118); or azadirachtin. (15) Octopaminergic agonists, forexample amitraz; (16) Site III electron transport inhibitors/site IIelectron transport inhibitors, for example hydramethylnon; acequinocyl;fluacrypyrim; or cyflumetofen and cyenopyrafen. (17) Electron transportinhibitors, for example site I electron transport inhibitors from thegroup of the METI acaricides, for example fenazaquin, fenpyroximate,pyrimidifen, pyridaben, tebufenpyrad, tolfenpyrad, and rotenone; orvoltage-dependent sodium channel blockers, for example indoxacarb andmetaflumizone. (18) Fatty acid biosynthesis inhibitors, for exampletetronic acid derivatives, for example spirodiclofen and spiromesifen;or tetramic acid derivatives, for example spirotetramat. (19) Neuronalinhibitors with unknown mechanism of action, for example bifenazate.(20) Ryanodin receptor effectors, for example diamides, for exampleflubendiamide, (R)-,(S)-3-chloro-N¹-{2-methyl-4-[1,2,2,2-tetrafluoro-1-(trifluoromethyl)-ethyl]phenyl}-N²-(1-methyl-2-methylsulphonylethyl)phthalamide,chlorantraniliprole (Rynaxypyr), or cyantraniliprole (Cyazypyr). (21)Further active ingredients with unknown mechanism of action, for exampleamidoflumet, benclothiaz, benzoximate, bromopropylate, buprofezin,chinomethionat, chlordimeform, chlorobenzilate, clothiazoben,cycloprene, dicofol, dicyclanil, fenoxacrim, fentrifanil, flubenzimine,flufenerim, flutenzin, gossyplure, japonilure, metoxadiazone, petroleum,potassium oleate, pyridalyl, sulfluramid, tetrasul, triarathene, orverbutin; or the following known active compounds:4-{[(6-bromopyrid-3-yl)methyl](2-fluoroethyl)amino}furan-2(5H)-one(known from WO 2007/115644),4-{[(6-fluoropyrid-3-yl)methyl](2,2-difluoroethyl)amino}furan-2(5H)-one(known from WO 2007/115644), 4-{[(2-chloro-1,3-thiazol-5-yl)methyl](2-fluoroethyl)amino}furan-2(5H)-one (known from WO 2007/115644),4-{[(6-chloropyrid-3-yl)methyl](2-fluoroethyl)amino}furan-2(5H)-one(known from WO 2007/115644),4-{[(6-chloropyrid-3-yl)methyl](2,2-difluoroethyl)amino}furan-2(5H)-one(known from WO 2007/115644), 4-{[(6-chloro-5-fluoropyrid-3-yl)methyl](methyl)amino}furan-2(5H)-one (known from WO 2007/115643),4-{[(5,6-dichloropyrid-3-yl)methyl](2-fluoroethyl)amino}furan-2(5H)-one(known from WO 2007/115646),4-{[(6-chloro-5-fluoropyrid-3-yl)methyl](cyclopropyl)amino}furan-2(5H)-on-e(known from WO 2007/115643),4-{[(6-chloropyrid-3-yl)methyl](cyclopropyl)amino}furan-2(5H)-one (knownfrom EP-A-0 539 588),4-{[(6-chloropyrid-3-yl)methyl](methyl)amino}furan-2(5H)-one (known fromEP-A-0 539 588),[(6-chloropyridin-3-yl)methyl](methyl)oxido-lambda⁴-sulphanylidenec-yanamide(known from WO 2007/149134),[1-(6-chloropyridin-3-yl)ethyl](methyl)oxido-lambda⁴-sulphanylidene-cyanamide(known from WO 2007/149134) and its diastereomers (A) and (B) (likewiseknown from WO 2007/149134),[(6-trifluoromethylpyridin-3-yl)methyl](methyl)oxido-lambda⁴-sulpha-nylidenecyanamide(known from WO 2007/095229), or[1-(6-trifluoromethylpyridin-3-yl)ethyl](methyl)oxidolambda⁴-sulph-anylidenecyanamide (known from WO 2007/149134) and itsdiastereomers (C) and (D), namely sulfoxaflor (likewise known from WO2007/149134).

The invention will now be described with reference to the followingexamples. It should be appreciated that these examples are not intendedto limit the scope of the claims to the invention, but are ratherintended to be exemplary of certain embodiments. Any variations in theexemplified methods that occur to the skilled artisan are intended tofall within the scope of the invention.

EXAMPLES Example 1. Domain Swap in Vip3D Mutant (10his-Vip3D-AAPF; P021)

One-hundred and twenty six amino acids in 10His-Vip3D-AAPF (P012) (SEQID NO: 6) were replaced with a carbohydrate binding module from a β-1,4mannanase (ManA) from Thermoanaerobacterium polysaccharolyticum(Caldanaerobius polysaccharolyticus). Accordingly, 137 amino acidscorresponding to a CBM from GenBank ID: AAD09354.1 (620 EGGVNMVSNPGFEDGLDSWQ DWQQDMSAVP EAAHNGALGL KIGGGKAAGG GQDIPLKPNT TYILGAWAKFDSKPAGTFDV VVQYHLKDAN NTYVQHILNF NETDWTYKQL LFTTPDVFGS TPQLALWKGDTSKANLYVDD VYLVEV 756)(SEQ ID NO:24) were inserted in place of aminoacids 542-667 of P021 (542 GSIEEDNLEP WKANNKNAYV DHTGGVNGTK ALYVHKDGGFSQFIGDKLKP KTEYVIQYTV KGKPSIHLKD ENTGYIHYED TNNNLKDYQT ITKRFTTGTDLKGVYLILKS QNGDEAWGDK FTILEI 667) (SEQ ID NO:6, aa 542-667). The designwas based on the atomic-resolution structure of Vip3D.

Bacillus thuringiensis codon tables were used for preparing the CBMportions of the chimeras. However, since expression of the chimericnucleotide sequences was carried out in E. coli, the codon table couldhave been an E. coli codon table or that of another prokaryote.

The chimeric domain-swap mutant (called Vip3Dd3to2ZEX) was determined tobe active (insecticidal) against black cutworm (BCW) (LC₅₀˜700 ng/cm²)and partially active toward fall armyworm (FAW) and western cornrootworm (WCR).

Example 2. Additional Domain III Swaps in P021

Based on the success of Vip3Dd3to2ZEX, eight other CBMs from eubacterialβ-1,4 mannanase enzymes were substituted for Vip3D Domain III (aminoacids 542-667) (Vip3Dd3) from the P021 sequence. The eight CBMs usedwere SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12,SEQ ID NO:13, SEQ ID NO:14, and SEQ ID NO:15. One domain swap (Vip3Dd3to 1WKy (SEQ ID NO:15) was insoluble after expression in E. coli, butthe remaining 7 killed 100% BCW larvae after 7 days when assayed as anE. coli soluble crude extract (Table 1).

TABLE 1 E. coli crude extract efficacy against black cutworm at 5 and 7days in a diet surface overlay assay. E. coli crude extract Day 5 Day 7Treatment Total# #Dead % Mortality Total# #Dead % Mortality Empty vectorcontrol 12 2 17% 12 3  25% Buffer control 12 0  0% 12 0  0% P021 12 12100%  12 12 100% Vip3Dd3to2ZEZ 12 12 100%  12 12 100% Vip3Dd3to2BGP 1211 92% 12 12 100% Vip3Dd3to1OF3 12 12 100%  12 12 100% Vip3Dd3to1PMH 1211 92% 12 12 100% Vip3Dd3to1WKY (insoluble) 12 0  0% 12 0  0%Vip3Dd3toCENC 12 10 83% 12 12 100% Vip3Dd3toGP21 12 0  0% 12 12 100%Vip3Dd3toPsHGF7 12 12 100%  12 12 100%

The soluble fraction of lysed E. coli expressing the various CBM Vip3DDomain III (P021) swaps is provided in FIG. 2. As shown, Vip3D3d to 1WKYdid not produce soluble protein. Thus, this fusion could not be testedreadily for efficacy.

Each of 2ZEX, 2ZEZ, 1OF3, 1PMH, 1WKY, and 2GBP are from solvedstructures which contain carbohydrate binding modules from Type B β-1,4mannanase enzymes (FIG. 3).

In addition to testing CBM swaps based on existing Protein Databankentries, three CBM fusions from other β-1,4 mannanase proteins weretested based on their BLAST similarity: CenC, gp21, and PsHGF7 (FIG. 4).

The first swap listed in Table 1 (Vip3Dd3to2ZEZ) is a CBM from the sameenzyme as that used in Vip3Dd3to2ZEX, GenBank ID: AAD09354.1. Thatparticular mannanase has two CBMs in tandem after the catalytic domain.2ZEX is the first CBM immediately followed by 2ZEZ. Despite apparentlyhaving the same substrate and being connected to the same enzyme, the2ZEZ CBM sequence shares 64% identity with 2ZEX. The 2ZEZ sequence is(756 GMDGWPDWGY PVSAVPEAAY GGTKGFKLSG GKQAGMGQKV ALKPNTTYIL GAWGKFTAKPGTYCDVIVQY HLKDANNTYV QNILRFTETD WTYKQVVFTT PDAFGSDPEF VLWKDDASNADFYADNITLV EV 899) (SEQ ID NO:25). Both 2ZEX and 2ZEZ have beenclassified into CBM family 16.

Provided below are the CBM sequences that were substituted in place ofamino acids 542 to 667 of P021.

2BGP GenBank: AAO31761.1 endo-b1,4-mannanase 5C[Cellvibrio japonicus]. CBM35 (SEQ ID NO: 28)(208 TAASASITAP AQLVGNVGEL QGAGSAVIWN VDVPVTGEYRINLTWSSPYS SKVNTLVMDG TALSYAFAEA TVPVTYVQTKTLSAGNHSFG VRVGSSDWGY MNVHSLKLEL LG 319)1OFE is from the C-terminal 176 amino acids of aThermotoga maritima endo β-1,4 mannanase. It hasbeen classified into CBM family 27. NP_229032 amino acids(SEQ ID NO: 26) (505 DFSSPEEVKN WWNSGTWQAE FGSPDIEWNG EVGNGALQLNVKLPGKSDWE EVRVARKFER LSECEILEYD IYIPNVEGLKGRLRPYAVLN PGWVKIGLDM NNANVESAEI ITFGGKEYRRFHVRIEFDRT AGVKELHIGV VGDHLRYDGP IFIDNVRLYKR 665)were cloned into Vip3D domain 3. 1PMH. CBM27-1. GenBank: AAC44232.1.Caldicellulosiruptor saccharolyticus β-1,4 mannanase. (SEQ ID NO: 27)(45 DFEDGTVMSF GEAWGDSLKC IKKVSVSQDL QRPGNKYALRLDVEFNPNNG WDQGDLGTWI GGVVEGQFDF TGYKSVEFEMFIPYDEFSKS QGGFAYKVVI NDGWKELGSE FNITANAGKKVKINGKDYTV IHKAFAIPED FRTKKRAQLV FQFAGQNSNY KGPIYLDNVR IRPE 218)1WKY. CBM59. β-1,4-mannanase.Caldicellulosiruptor saccharolyticus. (alkalinemannanase) (Amn5). GenBank: AAC44232.1; (SEQ ID NO: 32)(345 DFEESTQGWT GSSLSRGPWT VTEWSSKGNH SLKADIQMSSNSQHYLHVIQ NRSLQQNSRI QATVKHANWG SVGNGMTARLYVKTGHGYTW YSGSFVPING SSGTTLSLDL SNVQNLSQVREIGVQFQSES NSSGQTSIYI DNVIVE 490)CenC. carbohydrate-binding. CenC domain protein[Paenibacillus sp. JDR-2]. NCBI Reference Sequence: YP_003011283.1.;(SEQ ID NO: 30) (202 PGLEDGINNW QAWGEGFTAA SDMSHTGSAS LKVLLNNGGRQVVALQPGKS YKLGVWGKTA GTGTGTQTAT VMINYKKPEDDSSHTYGSFQ FGPDNSEFTY KEITFETPDD MAQEWGTQFV SIWSEGADQV YLDDFTLSEV 341)Gp21. Gp21 [Klebsiella pneumoniae subsp.pneumoniae HS11286]. NCBI Reference Sequence: YP_005220866.1.;(SEQ ID NO: 29) (69 PSFERGTEGY TGWSGIATVV TLQVPHLGTK AAKLAAGGSAGVGQKISFKK DRSYKIGIWA KQDPNTTIQS TDNTKFRVADGNGLIASKAY GPFTSNWQEV SWTWKATKDV LADVQFTAFL SAGAMYFDDF YVVDV 203)PsHGF7. carbohydrate binding domain protein[Paenibacillus sp. HGF7]. NCBI Reference Sequence: WP_009674454.1.(SEQ ID NO: 31) (66 PGFEDNLASW TNWGNTSSVT SPAFAGAKAA RIASGEGGAGQIIPGIPSGT TYVLSGHGSV SAGTDTAIVG VDCLDANNNVLAKNTLRFNQ TLYEFKSTAF TTVPGTAKLQ VYIYKNADSG ANAFLDDLSL VEV 195)

Schematics of the substitutions are provided in FIGS. 6A-6C.

Example 3. Activity Spectrum Test

All soluble constructs were purified via nickel affinity chromatography.The proteins exchanged into phosphate buffered saline (PBS) and testedvia diet overlay against a variety of lepidopteran species at the L1-L2stage (n=12). Two doses were tested: 32 μg/cm² and 1 μg/cm². Controlswere purified P021, which is 10-histidine tagged Vip3D with amino acidsAAPF substituted at the active site (SEQ ID NO:6) and a buffer control(PBS). The activity of each of the constructs was tested againstEuropean Corn Borer (ECB), Corn Earworm (CEM), Black Cutworm (BCW), andFall Armyworm (FAW). The results of the activity tests are provided inTable 2, below.

TABLE 2 Activity of P012 swaps against European Corn Borer (ECB), CornEarworm (CEM), Black Cutworm (BCW), and Fall Armyworm (FAW). 144 hourassay % Mortality % Mortality Diet overlay 32 μg/cm² 1 μg/cm² EuropeanCorn Borer P021 (HisVip3D_AAPF) 100 8 Vip3Dd3_to_2ZEX 17 0Vip3Dd3_to_2ZEZ 0 0 Vip3Dd3_to_1OFE 0 0 Vip3Dd3_to_1PMH 0 8Vip3Dd3_to_2BGP 0 0 Vip3Dd3_to_GP21 67 0 Vip3Dd3_to_CENC 0 0Vip3Dd3_to_PSHGF7 0 0 PBS 8 0 Black Cutworm P021 (HisVip3D_AAPF) 100 100Vip3Dd3_to_2ZEX 100 92 Vip3Dd3_to_2ZEZ 100 83 Vip3Dd3_to_1OFE 92 0Vip3Dd3_to_1PMH 92 0 Vip3Dd3_to_2BGP 92 25 Vip3Dd3_to_GP21 100 100Vip3Dd3_to_CENC 100 0 Vip3Dd3_to_PSHGF7 100 92 PBS 0 0 Corn Earworm P021(HisVip3D_AAPF) 100 83 Vip3Dd3_to_2ZEX 75 100 Vip3Dd3_to_2ZEZ 92 92Vip3Dd3_to_1OFE 0 67 Vip3Dd3_to_1PMH 25 8 Vip3Dd3_to_2BGP 33 83Vip3Dd3_to_GP21 100 92 Vip3Dd3_to_CENC 0 0 Vip3Dd3_to_PSHGF7 83 33 PBS 88 Fall Armyworm P021(HisVip3D_AAPF) 100 100 Vip3Dd3_to_2ZEX 100 92Vip3Dd3_to_2ZEZ 100 67 Vip3Dd3_to_1OFE 33 8 Vip3Dd3_to_1PMH 42 17Vip3Dd3_to_2BGP 25 17 Vip3Dd3_to_GP21 100 100 Vip3Dd3_to_CENC 33 8Vip3Dd3_to_PSHGF7 100 83 PBS 0 8

The results of swapping β-1,4 mannanase Type B CBM sequences in place ofVip3D Domain III are clear. Broad spectrum Lepidoptera control isobserved using purified proteins. Certain chimeras were more or lesstoxic to any given insect at a particular dose. For exampleVip3Dd3_to_GP21 seems to be one of the most toxic of the fusions againstthe particular insects tested; however, it is less toxic against CEWthan is Vip3Dd3_to_2ZEX. The chimeras are likely to be useful incontrolling multiple lepidopteran and other agricultural pests.

Example 4. Vip3A Swaps

The same CBM domains that were swapped with Domain III of P021 asdescribed in Examples 1 and 2 were swapped with Domain III of Vip3Apolypeptides. The CBM domains were cloned onto the Vip3A scaffold at thesame junction points (e.g., amino acids 541 and 668) as in Vip3D P021.The Vip3A proteins did not have the AAPF change in the activation siteand did not contain an N-terminal histidine tag as in P021. Due to alack of a single-column purification step, E. coli soluble crude extractdiet overlays were used for the spectrum test. These constructsexpressed well in crude extracts and as such these assays were ahigh-dose test. An empty-vector E. coli crude extract was included as acontrol. The results are provided in Table 3, below.

The SDS-PAGE gel shows that nearly all proteins were highly soluble,with the exception of the 2ZEX and 1OFE chimeras (FIG. 5). These twoconstructs did have some soluble protein expression and activity asshown in Table 3 (below).

TABLE 3 Activity of Vip3A Domain III swaps against European Corn Borer(ECB), Corn Earworm (CEM), Black Cutworm (BCW), and Fall Armyworm (FAW).144 hour assay % Mortality Diet overlay Crude Extract European CornBorer Empty Vector  0% Vip3Ad3_to_2ZEX  0% Vip3Ad3_to_2ZEZ  25%Vip3Ad3_to_1OFE  0% Vip3Ad3_to_1PMH  0% Vip3Ad3_to_2BGP  0%Vip3Ad3_to_GP21  33% Vip3Ad3_to_CENC  0% Vip3Ad3_to_PSHGF7  0% PBS  0%Black Cutworm Empty Vector  0% Vip3Ad3_to_2ZEX 100% Vip3Ad3_to_2ZEZ 100%Vip3Ad3_to_1OFE 100% Vip3Ad3_to_1PMH 100% Vip3Ad3_to_2BGP 100%Vip3Ad3_to_GP21 100% Vip3Ad3_to_CENC 100% Vip3Ad3_to_PSHGF7 100% PBS  0%Corn Earworm Empty Vector  0% Vip3Ad3_to_2ZEX  75% Vip3Ad3_to_2ZEZ 100%Vip3Ad3_to_1OFE  0% Vip3Ad3_to_1PMH  75% Vip3Ad3_to_2BGP 100%Vip3Ad3_to_GP21 100% Vip3Ad3_to_CENC  83% Vip3Ad3_to_PSHGF7 100% PBS  0%Fall Armyworm Empty Vector  0% Vip3Ad3_to_2ZEX 100% Vip3Ad3_to_2ZEZ 100%Vip3Ad3_to_1OFE  8% Vip3Ad3_to_1PMH 100% Vip3Ad3_to_2BGP 100%Vip3Ad3_to_GP21 100% Vip3Ad3_to_CENC 100% Vip3Ad3_to_PSHGF7 100% PBS  0%

The results show that the same CBM swaps are effective for broadlepidopteran control in Vip3A. The overall pattern in ECB is similar tothe high-dose Vip3D swaps with 2ZEZ and GP21 being most active. Theother 3 insect species tested were also highly susceptible to thesefusions. However, the 10OFE swap was only toxic to BCW. Gel analysisindicates that 1OFE was expressed at much lower levels than most of theother proteins (FIG. 5), which could account for the aberrantinactivity. The 2ZEX fusion was also expressed at lower levels butshowed high activity to all insect larvae except ECB. The data in theabove examples show that Type B Carbohydrate Binding Modules from β 1, 4mannanase enzymes can be swapped in place of Domain III of Vip3polypeptides to generate active toxins. These toxins showed differentialactivity towards the four Lepidoptera species tested with the majoritykilling FAW, CEW, and BCW. ECB was effectively targeted by two chimeras.Vip3D and Vip3A both served as good scaffolds for these designs andgenerated differential activities. The insects tested are exemplary ofthe types that may be controlled by the constructs of this invention.Further, the specific CBMs that are swapped for Domain III of Vip3polypeptides are also exemplary of the types of CBM domains that can beused with this invention. Thus, any β-1,4 mannanase CBM, in particularany β-1,4 mannanase Type B CBM, may be useful with this invention whenintroduced into a Vip3 polypeptide or exchanged with Domain III of aVip3 polypeptide. Further, in some embodiments, any CBM having at least70% homology or identity to any one of the amino acid sequence of SEQ IDNOs:24 to 32 may be useful with this invention when introduced into aVip3 polypeptide or exchanged with Domain III of a Vip3 polypeptide.

Notably, in addition to controlling insects directly with thecompositions described herein, the constructs can also be expressed intransgenic plants providing additional tools to the farmer for controlof plant pests.

The above examples clearly illustrate the advantages of the invention.Although the present invention has been described with reference tospecific details of certain embodiments thereof, it is not intended thatsuch details should be regarded as limitations upon the scope of theinvention except as and to the extent that they are included in theaccompanying claims.

That which is claimed is:
 1. A modified Vip3 polypeptide comprising aheterologous carbohydrate binding module (CBM), wherein the CBM is froma bacterial β-1,4-mannanase, wherein the heterologous CBM is substitutedfor all or a portion of Domain III of a Vip3 polypeptide, and whereinthe modified Vip3 polypeptide is pesticidal against an insect.
 2. Themodified Vip3 polypeptide of claim 1, wherein the Vip3 polypeptidecomprises all or a portion of SEQ ID NO:1.
 3. The modified Vip3polypeptide of claim 1, wherein all or a portion of Domain III comprisesamino acids 542 to 667 of SEQ ID NO:1.
 4. The modified Vip3 polypeptideof claim 1, wherein the heterologous CBM comprises a metal binding site.5. The modified Vip3 polypeptide of claim 1, wherein the modified Vip3polypeptide is pesticidal against a lepidopteran insect.
 6. The modifiedVip3 polypeptide of claim 1, wherein the modified Vip3 polypeptide ispesticidal against at least one of Ostrinia nubilalis (European cornborer), Plutella xylostella (diamondback moth), Spodoptera frugiperda(fall armyworm), Agrotis ipsilon (black cutworm), Agrotis orthogonia(pale western cutworm), Striacosta albicosta (western bean cutworm),Helicoverpa zea (corn earworm), Heliothis virescens (tobacco budworm),Spodoptera exigua (beet armyworm), Helicoverpa punctigera (nativebudworm), Helicoverpa armigera (cotton bollworm), Manduca sexta (tobaccohornworm), Trichoplusia ni (cabbage looper), Pectinophora gossypiella(pink bollworm), Diatraea grandiosella (southwestern corn borer),Diatraea saccharalis (sugarcane borer), Elasmopalpus lignosellus (lessercornstalk borer), Psuedoplusia includens (soybean looper), Anticarsiagemmatalis (velvetbean caterpillar), Plathypena scabra (greencloverworm), Homoeosoma electellum (sunflower head moth), and Cochylishospes (banded sunflower moth), or any combination thereof.
 7. Acomposition comprising the modified Vip3 polypeptide of claim 1 in anagriculturally acceptable carrier.
 8. The modified Vip3 polypeptide ofclaim 1, wherein the heterologous CBM is SEQ ID NO:24.
 9. The modifiedVip3 polypeptide of claim 1, wherein the modified Vip3 polypeptidecomprises SEQ ID NO:7.